Long-Acting Formulations of Insulin

ABSTRACT

The application relates to an aqueous pharmaceutical formulation comprising 200-1000 U/mL [equimolar to 200-1000 IU human insulin] of insulin glargine.

This application is a continuation application of U.S. Ser. No.13/110,568, filed May 18, 2011, which claims benefit to U.S. ProvisionalAppln. Nos. 61/411,608, filed Nov. 9, 2010 and 61/429,936, filed Jan. 5,2011.

The application relates to an aqueous pharmaceutical formulationcomprising 200-1000 U/mL [equimolar to 200-1000 IU human insulin] ofinsulin glargine, and its use.

Insulin glargine is 31^(B)-32^(B)-Di-Arg human insulin, an analogue ofhuman insulin, with further substitution of asparagine in position A21by glycine.

Lantus® is an insulin product containing insulin glargine providing 24hour basal insulin supply after single dose subcutaneous injection.

The glucodynamic effect of Lantus® is distinguished from other currentlymarketed insulin products by virtue of a delayed and predictableabsorption of insulin glargine from the subcutaneous injection siteresulting in a smooth, 24 hour time-concentration and action profilewithout a definite peak. Lantus® was developed to meet the medical needfor a long-acting insulin product that can be administered as a singledaily injection to yield normal or near-normal blood glucose controlwith a basal insulin profile that is as smooth as possible over a24-hour period. Such a preparation provides good control of bloodglucose all day, while minimizing the tendency to produce hypoglycemiaseen with other insulin preparations with a more definite “peak” effect.

A considerable number of patients, in particular those with increasedinsulin resistance due to obesity, use large doses to control bloodglucose. For example, a dose of 100 U requires injection of 1 mL Lantus®U100, which may confer some discomfort; each mL Lantus® U100 contains100 U (3.6378 mg) insulin glargine. To reduce the volume of injection, aformulation containing 300 U insulin glargine per mL has been developed.Although the invention is not limited to an insulin glargine U 300formulation, the clinical studies described herein were performed withan insulin glargine U 300 formulation; each mL insulin glargine U300contains 300 U (10.9134 mg) insulin glargine. This formulation wouldallow patients to inject the same number of units of insulin glargine atone third the volume of injection.

Both insulin glargine formulations, U100 and U300, were expected toprovide the same insulin exposure and the same effectiveness, i.e. timeprofiles.

FIGURES

The figures below effectively show the surprising and unexpecteddifferences in exposure (PK) and activity (PD) between Lantus U100 undLantus U300 formulations (insulin glargine U100 und insulin glargineU300 formulations) after the same s.c. dose given to healthy subjects,at the same time as blood glucose (PD) was constant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Glucose Infusion Rate (GIR) Lantus U100.

FIG. 2: Glucose Infusion Rate (GIR) Lantus U300.

FIGS. 3A and 3B: Serum Insulin Concentrations; Lantus U100 (3A) and U300(3B).

FIGS. 4A and 4B: Blood Glucose U100; (4A) replicates=1, (4B)replicates=2.

FIGS. 5A and 5B: Blood Glucose U300; (5A) replicates=1, (5B)replicates=2.

FIGS. 6A, 6B and 6C: Results of a randomized, 4-sequence, cross-over,double-blind, dose response study of 0.4, 0.6 and 0.9 U/kg HOE-901-U300(insulin glargine U300) compared to 0.4 U/kg Lantus® U100 (insulinglargine U100) in patients with diabetes mellitus type 1 using theeuglycemic clamp technique. FIG. 6A: insulin glargine concentration(mU/L), FIG. 6B: blood glucose (BG, mg/dL), FIG. 6C: glucose infusionrate (GIR, mg·kg⁻¹·min⁻¹). The curves display LOWESS smoothed averagesof all data points of all subjects (population averages); LOWESS is adata analysis technique for producing a “smooth” set of values from atime series which has been contaminated with noise, or from a scatterplot with a “noisy” relationship between the 2 variables.

FIG. 7: Glucose infusion rate (GIR, mg·kg⁻¹·min⁻¹). The curves displayLOWESS smoothed averages of all data points of all subjects (populationaverages); LOWESS is a data analysis technique for producing a “smooth”set of values from a time series which has been contaminated with noise,or from a scatter plot with a “noisy” relationship between the 2variables.

FIG. 7: Legend: Profiles 1 to 3 (from top to bottom).

Results of a randomized, double-blind, parallel group dose responsestudy of 0.4, 0.6 and 1.2 U/kg Lantus® U100 (insulin glargine U100) inpatients with diabetes mellitus type 1 using the euglycemic clamptechnique.

FIG. 7: Legend: Profiles 4 to 7 (from top to bottom).

Results of a randomized, 4-sequence, cross-over, double-blind, doseresponse study of 0.4, 0.6 and 0.9 U/kg HOE-901-U300 (insulin glargineU300) compared to 0.4 U/kg Lantus® U100 (insulin glargine U100) inpatients with diabetes mellitus type 1 using the euglycemic clamptechnique.

FIGS. 8A, 8B, 8C, 8D and 8E: Optical microscope pictures of precipitatesof insulin glargine formulations with increasing concentrations:

FIG. 8A: 100 U/mL, FIG. 8B: 300 U/mL, FIG. 8C: 500 U/mL, FIG. 8D: 700U/mL and FIG. 8E: 1000 U/mL,

with the magnitude of 100× and including the maximum diameters.

All precipitations are performed with 60 U of insulin glargine.

FIG. 9: Time-action profile of insulin glargine U-100 vs. U-300 innormoglycemic dogs.

DETAILED DESCRIPTION

Exposure and activity of insulin glargine U300, the test (T) medication,was tested in non-diabetic healthy subjects in euglycemic clamps forequivalence in exposure and activity to Lantus U100, the approvedreference (R) product. To account for the long duration of action ofinsulin glargine after subcutaneous administration 30 hours wereselected. Exposure was assessed from insulin glargine concentration timeprofiles after subcutaneous administration while activity wassimultaneously assessed as glucose utilization per unit insulin.

A replicate design allowed limiting the number of subjects for assessingbioequivalence and variability as recommended by the FDA guideline“Guidance for Industry, Statistical Approaches to EstablishingBioequivalence”.

The respective clinical study was expected to establish equivalence inexposure and activity.

A dose of 0.4 U/kg was selected for this study; it corresponds to theaverage basal insulin dose in patients. In non-diabetic healthy subjectsthis dose produces a sizeable elevation in plasma insulin concentrationand a lasting glucose lowering effect that can be quantified ineuglycemic clamp settings.

The replicate design favored by guidelines requires two replicate singledose injections of either IP (R: Lantus® U100, T: insulin glargine U300)in predefined four way cross-over sequences (RTTR or TRRT) as allottedby the randomization plan. This was executed in Periods (P) 1-4 at fourdifferent days. As a result, each subject received two replicate singlesubcutaneous doses of 0.4 U/kg Lantus® U100 (R) and insulin glargineU300 (T), alternating between two opposite sites of the periumbilicalarea.

A washout period of 4 to 18 days separated each dosing day. The lengthof the wash-out period varied individually allowing both the participantand the Investigator to adjust to their needs. By experience, 4 dayscomprise a minimum period for recovery, enabling 1 clamp per week for aparticipant, while 18 days represent a break of 3 weeks between clampdays, allowing subjects more freedom to fulfill non-study relatedobligations.

Prior to the euglycemic clamp visits, at SCR (screening visit), subjectshave been screened for eligibility, and in EOS (end-of-study) visitsubjects have come in for a final examination to ensure normal healthstatus. Screening and P1 have not be separated by more than 21 days,while the EOS visits occurred no earlier than the same week day as Day 1of P4 the following week, i.e. after an additional 4 days, and no laterthan a fortnight after Day 2 of P4, i.e. after an additional 14 days.

This has been a single dose study with in total 4 replicateadministrations. The effect of the IPs was to last about 24 hours, whichis why the subjects have been confined to the institute for 2 days.Subjects have been exposed to treatment 4 times.

The primary objective of the study was to assess the averagebioequivalence (ABE) of Lantus® U100 (commercial formulation) andinsulin glargine U300 in bioavailability (exposure) and bioefficacy(activity) using the euglycemic clamp technique.

The secondary objective of the study was to assess safety andtolerability of insulin glargine U300.

As mentioned above, both insulin glargine formulations, U100 and U300,were expected to provide the same insulin exposure and the sameeffectiveness. However, surprisingly insulin exposure and effectivenesswere shown to be not the same. Insulin glargine U 100 and insulinglargine U 300 are not equivalent in bio-availability (exposure) andbio-efficacy (activity). Exposure and activity after administration ofinsulin glargine U300 were less by about 40% as compared to exposure andactivity after administration of the same amount (0.4 U/kg) from insulinglargine U100.

Insulin glargine U300 did, however, show an even flatter PK (exposure)and PD (activity) profile than insulin glargine U100, as would bedesired for a basal insulin. These surprising and unexpected differencesin exposure and activity between insulin glargine U100 and insulinglargine U300 formulations after the same s.c. dose to healthy subjectsare effectively shown in the figures below. Of note, at the same timeblood glucose was constant.

The blood glucose lowering effect of insulin glargine was additionallyevaluated in healthy, normoglycemic Beagle dogs. With increasing insulinglargine concentration the mean time of action increased from 6.8 h(U100) to 7.69 h (U300), respectively.

By increasing the glargine concentration from 100 to 300 U/mL the bloodglucose decreasing time-action profile was changed towards a flatter andprolonged activity in the dog. The current data in dogs is consistentwith data in humans showing that higher drug concentrations of insulinglargine are positively correlated with profile and longer duration ofaction.

Additionally, the precipitates of insulin glargine formulations havingconcentrations of 100 U/mL, 300 U/mL, 500 U/mL 700 U/mL and 1000 U/mLhave been investigated by microscopy. These investigations revealeddifferences in the precipitations characteristics, leading to remarkablebigger particles with increasing concentrations.

Furthermore, the influence of the higher concentrations of insulinglargine formulations with regard to dissolution properties areinvestigated by using an in-vitro test system. To do so, precipitationstudies were performed using a phosphate buffer with a pH of 7.4,simulating the in-vivo conditions.

The supernatant of the precipitated insulin was investigated using HPLCtechnique to determine the insulin glargine content.

The present invention is not limited to an insulin glargine U 300formulation and is effective with other higher concentrated formulationsof insulin glargine as outlined in detail in the specification, theclinical studies described herein were performed with an insulinglargine U 300 formulation.

Specifically, the insulin glargine formulations of the present inventionexhibit a flatter PK (exposure) and flatter PD (activity) profile thaninsulin glargine U100 and surprisingly act as improved basal insulinscompared to U100 glargine insulin and therefore impart extended durationof exposure and reduce the incidence of hypoglycemia in the treatment ofType I and Type II diabetes, for example.

1 mL of insulin glargine U 300 formulation contains 10.913 mg21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg human insulin [equimolar to 300IU human insulin], 90 μg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, HCland NaOH ad pH 4.0; specific gravity 1.006 g/mL

However, variations with regard to the kind of excipients and theirconcentrations are possible.

The pharmaceutical formulation of the present invention contains200-1000 U/mL of insulin glargine [equimolar to 200-1000 IU humaninsulin], preferably 250-500 U/mL of insulin glargine [equimolar to250-500 IU human insulin], more preferred 270-330 U/mL of insulinglargine [equimolar to 270-330 IU human insulin], and even morepreferred 300 U/mL of insulin glargine [equimolar to 300 IU humaninsulin].

In one embodiment, the present invention is directed to an aqueouspharmaceutical formulation comprising insulin glargine in the range of200-1000 U/mL [equimolar to 200-1000 IU human insulin], preferably 200U/ml to 650 U/mL, still preferably 700 U/mL to 1000 U/ml, morepreferably 270-330 U/mL and most preferably in a concentration of 300U/mL.

In one embodiment, the present invention is directed to an aqueousformulation comprising 200-1000 U/mL [equimolar to 200 to 1000/U humaninsulin] of insulin glargine, with the proviso that the concentration ofinsulin in said formulation is not 684 U/ml of insulin glargine.

In another embodiment, the pharmaceutical formulation of the presentinvention contains 200 U/mL of insulin glargine (equimolar to 200 IUhuman insulin] or 300 U/mL of insulin glargine [equimolar to 300 IUhuman insulin] or 400 U/mL of insulin glargine [equimolar to 400 IUhuman insulin] or 500 U/mL of insulin glargine [equimolar to 500 IUhuman insulin] or 600 U/mL of insulin glargine [equimolar to 600 IUhuman insulin] or 700 U/mL of insulin glargine [equimolar to 700 IUhuman insulin] or 800 U/mL of insulin glargine [equimolar to 800 IUhuman insulin] or 900 U/mL of insulin glargine [equimolar to 900 IUhuman insulin] or 1000 U/mL of insulin glargine [equimolar to 1000 IUhuman insulin].

Surfactants can be added to pharmaceutical formulation, for example,inter alia, non-ionic surfactants. In particular, pharmaceuticallycustomary surfactants are preferred, such as, for example:

partial and fatty acid esters and ethers of polyhydric alcohols such asof glycerol, sorbitol and the like (Span®, Tween®, in particular Tween®20 and Tween® 80, Myrj®, Brij®), Cremophor® or poloxamers. Thesurfactants are present in the pharmaceutical composition in aconcentration of 5-200 μg/ml, preferably of 5-120 μg/ml and particularlypreferably of 20-75 μg/ml.

The formulation of the present invention can additionally containpreservatives (e.g. phenol, m-cresol, p-cresol, parabens), isotonicagents (e.g. mannitol, sorbitol, lactose, dextrose, trehalose, sodiumchloride, glycerol), buffer substances, salts, acids and alkalis andalso further excipients. These substances can in each case be presentindividually or alternatively as mixtures.

Glycerol, dextrose, lactose, sorbitol and mannitol can be present in thepharmaceutical preparation in a concentration of 100-250 mM, NaCl in aconcentration of up to 150 mM. Buffer substances, such as, for example,phosphate, acetate, citrate, arginine, glycylglycine or TRIS (i.e.2-amino-2-hydroxymethyl-1,3-propanediol) buffer and corresponding salts,are present in a concentration of 5-250 mM, preferably 10-100 mM.Further excipients can be, inter alia, salts or arginine.

The zinc concentration of the formulation of the present invention is inthe range of the concentration which is reached by the presence of0-1000 μg/mL, preferably 20-400 μg/mL zinc, most preferably 90 μg/mL.However, the zinc may be present in form of zinc chloride, but the saltis not limited to be zinc chloride.

In the pharmaceutical formulation glycerol and/or mannitol can bepresent in a concentration of 100-250 mmol/L, and/or NaCl is preferablypresent in a concentration of up to 150 mmol/L.

In the pharmaceutical formulation a buffer substance can be present in aconcentration of 5-250 mmol/L.

A further subject of the invention is a pharmaceutical insulinformulation which contains further additives such as, for example, saltswhich delay the release of insulin. Mixtures of such delayed-releaseinsulins with formulations described above are included therein.

For producing the formulations of the present invention the ingredientsare dissolved in water and the pH is adjusted by using HCl and/or NaOH;and likewise by methods known in the art. Likewise, a further subject ofthe invention is directed to the use of such formulations for thetreatment of diabetes mellitus.

A further subject of the invention is directed to the use or theaddition of surfactants as stabilizers during the process for theproduction of insulin, insulin analogs or insulin derivatives or theirpreparations.

The invention further relates to a formulation as described above whichadditionally comprises also a glucagon-like peptide-1 (GLP1) or ananalogue or derivative thereof, or exendin-3 or -4 or an analogue orderivative thereof, preferably exendin-4.

The invention further relates to a formulation as described above inwhich an analogue of exendin-4 is selected from a group comprising

H-desPro³⁶-exendin-4-Lys₆-NH₂,H-des(Pro^(36,37))-exendin-4-Lys₄-NH₂ andH-des(Pro^(36,37))-exendin-4-Lys₅-NH₂,or a pharmacologically tolerable salt thereof.

The invention further relates to a formulation as described above inwhich an analogue of exendin-4 is selected from a group comprisingdesPro³⁶ [Asp²⁸]exendin-4 (1-39), desPro³⁶ [IsoAsp²⁸]exendin-4 (1-39),desPro³⁶ [Met(O)¹⁴, Asp²⁸]exendin-4 (1-39), desPro³⁶ [Met(O)¹⁴,IsoAsp²⁸]exendin-4 (1-39), desPro³⁶ [Trp(O₂)²⁵, Asp²⁸]exendin-2 (1-39),desPro³⁶ [Trp(O₂)²⁵, IsoAsp²⁸]exendin-2 (1-39), desPro³⁶[Met(O)¹⁴Trp(O₂)²⁵, Asp²⁸]exendin-4 (1-39) and desPro³⁶[Met(O)¹⁴Trp(O₂)²⁵, IsoAsp²⁸]exendin-4 (1-39), or a pharmacologicallytolerable salt thereof.

The invention further relates to a formulation as described in thepreceding paragraph, in which the peptide-Lys₆-NH₂ is attached to the Ctermini of the analogues of exendin-4.

The invention further relates to a formulation as described above inwhich an analogue of exendin-4 is selected from a group comprising

H-(Lys)₆-des Pro³⁶ [Asp²⁸]exendin-4(1-39)-Lys₆-NH₂des Asp²⁸Pro³⁶, Pro³⁷, Pro³⁸ exendin-4(1-39)-NH₂,H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸ [Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅ des Pro³⁶, Pro³⁷, Pro³⁸ [Asp²⁸]exendin-4(1-39)-NH₂,des Pro³⁶, Pro³⁷, Pro³⁸ [Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸ [Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸ [Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-des Pro [Trp(O₂)²⁵, Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,H-des Asp²⁸ Pro³⁶, Pro³⁷, Pro³⁸ [Trp(O₂)²⁵]exendin-4(1-39)-NH₂,H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸ [Trp(O₂)²⁵, Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸ [Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,des Pro³⁶, Pro³⁷, Pro³⁸ [Trp(O₂)²⁵, Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸ [Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸ [Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-des Pro³⁶ [Met(O)¹⁴, Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,des Met(O)¹⁴ Asp²⁸ Pro³⁶, Pro³⁷, Pro³⁸ exendin-4(1-39)-NH₂,H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Asp²⁸]exendin-4(1-39)-NH₂,des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,H-Asn-(Glu)₅ des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-des Pro³⁶ [Met(O)¹⁴, Trp(O₂)²⁵, Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,des Asp²⁸ Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Trp(O₂)²⁵]exendin-4(1-39)-NH₂,H-(Lys)₆-des Pro³⁶′Pro³⁷, Pro³⁸ [Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Asp²⁸]exendin-4(1-39)-NH₂,des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸ [Met(O)¹⁴, Trp(O₂)²⁵, Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂, H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro[Met(O)¹⁴, Trp(O₂)²⁵, Asp²⁸] exendin-4(1-39)-(Lys)₆-NH₂,or a pharmacologically tolerable salt thereof.

The invention further relates to a formulation as described above whichadditionally comprises Arg³⁴, Lys²⁶(N^(ε)(γ-glutamyl(N^(α)-hexadecanoyl))) GLP-1 (7-37) [liraglutide] or apharmacologically tolerable salt thereof.

Additionally, the formulation of the present invention can also comprisean analogue of exendin-4, such, for example, lixisentatide, exenatideand liraglutide. These exendin-4 analogues are present in theformulation in the range of 0.1 μg to 10 μg per U insulin glargine,preferably 0.2 to 1 μg per U insulin glargine, and more preferably 0.25μg to 0.7 μg per U insulin glargine. Lixisenatide is preferred.

Additionally, the aqueous pharmaceutical formulation can comprise one ormore excipients selected from a group comprising zinc, m-cresol,glycerol, polysorbate 20 and sodium. Specifically, the aqueouspharmaceutical formulation can comprise 90 μg/mL zinc, 2.7 mg/mLm-cresol and 20 mg/ml glycerol 85%. Optionally, the aqueouspharmaceutical formulation can comprise 20 μg/mL polysorbate 20.

The pH of the aqueous pharmaceutical formulation is between 3.4 and 4.6,preferably 4 or 4.5.

The present invention is directed to a method of treating Type I andType II Diabetes Mellitus comprising administering to said patient theaqueous pharmaceutical composition of the present invention to adiabetic patient. Preferred among the various disclosed concentrationranges is a concentration of 300 U/mL and the preferred insulin analogueis insulin glargine. Further the aqueous pharmaceutical formulation alsocan comprise zinc, m-cresol, glycerol, polysorbate 20 and sodium andmixtures thereof in the ranges disclosed herein in relation to theaqueous pharmaceutical formulation of the present invention. In apreferred embodiment the aqueous pharmaceutical formulation alsocomprises 0.1 μg to 10 μg lixisenatide per U insulin glargine.

The insulin is administered preferably once daily but can beadministered twice daily as needed. Dosage requirements are a functionof the needs of the individual patient determined by the achievement ofnormal or acceptable blood glucose levels.

The present invention is also directed to a method of extending theduration of exposure of insulin glargine in the treatment of Type I andType II Diabetes Mellitus in a patient comprising administering to saidpatient the aqueous pharmaceutical formulation of the present invention.Preferred among the various disclosed concentration ranges is aconcentration of 300 U/mL. Further the aqueous pharmaceuticalformulation also can comprise zinc, m-cresol, glycerol, polysorbate 20and sodium and mixtures thereof in the ranges disclosed herein inrelation to the aqueous pharmaceutical formulation of the presentinvention.

In a preferred embodiment the aqueous pharmaceutical formulation alsocomprises 0.1 μg to 10 μg lixisenatide per U insulin glargine.

The present invention is also directed to a method of reducing theincidence of hypoglycaemia in the treatment of Type I and Type IIDiabetes Mellitus in a patient with insulin glargine comprisingadministering to said patient the aqueous pharmaceutical formulation ofthe present invention. Preferred among the various disclosedconcentration ranges is a concentration of 300 U/mL. Further the aqueouspharmaceutical formulation also can comprise zinc, m-cresol, glycerol,polysorbate 20 and sodium and mixtures thereof in the ranges disclosedherein in relation to the aqueous pharmaceutical formulation of thepresent invention. In a preferred embodiment the aqueous pharmaceuticalformulation also comprises 0.1 μg to 10 μg lixisenatide per U insulinglargine.

The present invention is also directed to a method of providing apeakless long acting basal insulin in the treatment of Type I and TypeII Diabetes Mellitus in a patient with comprising administering to saidpatient the aqueous pharmaceutical formulation of the present invention.Preferred among the various disclosed concentration ranges is aconcentration of 300 U/mL. Further the aqueous pharmaceuticalformulation also can comprise zinc, m-cresol, glycerol, polysorbate 20and sodium and mixtures thereof in the ranges disclosed herein inrelation to the aqueous pharmaceutical formulation of the presentinvention. In a preferred embodiment the aqueous pharmaceuticalformulation also comprises 0.1 μg to 10 μg lixisenatide per U insulinglargine.

Use of an aqueous formulation according to any of the foregoing items inthe treatment of Type 1 Diabetes Mellitus and Type 2 Diabetes Mellitus.

The application is described below with the aid of some examples, whichare in no way intended to act restrictively.

Example 1: Description of the Protocol

This study was a single center, randomized, controlled, single-blind,four-period, 2-treatment, 2-sequence crossover study in healthy subjectswith six visits:

Visit 1: Screening (SCR)

Visit 2 to 5, Period (P) 1-4: Treatment, euglycemic clamp periodVisit 6: End-of-study (EOS)Subjects received single subcutaneous doses of 0.4 U/kg insulin glargineU100 and insulin glargine U300 alternatingly injected into two oppositesites of the periumbilical area (left, right, left, right) at fourdifferent days. The study medication was administered with a replicateof treatment R and T in 2 sequences, RTTR or TRRT at P1 to P4. A washoutperiod of 4 to 18 days was separated each dosing day.R: 0.4 U/kg body weight insulin glargine U100 (commercial formulation;Reference)T: 0.4 U/kg body weight insulin glargine U300 (Test)P1 must take place no more than 3 to 21 days after SCR. EOS visit musttake place between 4 to 14 days after P4.

During P1 to P4, subjects have been connected to a Biostator formeasurement of blood glucose and adjustment of glucose infusion rate.Blood glucose levels and glucose infusion rate (GIR) have been monitoredfor 90 minutes (baseline period) before subcutaneous injection of thestudy medication and for 30 hours after study medication administration.Infusion of 20% glucose solution commenced to maintain blood glucoselevels at 5% below the individual fasting blood glucose level,determined as the mean of the 3 fasting blood glucose values measured60, 30 and 5 minutes before study medication administration. Profiles ofGIR have been obtained. Blood samples have been taken at predeterminedtimes during the euglycemic clamp period for determination of seruminsulin glargine concentrations. With the exception of tap water,subjects have been fasting during the glucose clamp period.

The duration of this study for an individual was expected to be up to 13weeks between SCR and EOS visit.

The protocol was submitted to independent ethics committees and/orinstitutional review boards for review and written approval. Theprotocol complied with recommendations of the 18th World Health Congress(Helsinki, 1964) and all applicable amendments. The protocol alsocomplied with the laws and regulations, as well as any applicableguidelines, of Germany, where the study was conducted. Informed consentwas obtained prior to the conduct of any study-related procedures.

Example 2: Selection of Subjects

Twenty four (24) healthy subjects were planned to be treated in order tohave 20 completers.

Subjects meeting all of the following criteria have been considered forenrollment into the study:

Demography

-   -   Subjects of either gender between 18 and 50 years of age;    -   Body weight between 50 kg and 110 kg and Body Mass Index between        18 and 28 kg/m²;

Health Status

-   -   Certified as healthy following a comprehensive clinical        assessment (detailed medical history and complete physical        examination);    -   Non-smoker for at least 3 months;    -   12-lead electrocardiogram, and vital signs unless the        Investigator considers an abnormality to be clinically        irrelevant        -   Normal vital signs after 5 minutes resting in supine            position:            -   95 mmHg≤systolic blood pressure≤140 mmHg;            -   45 mmHg≤diastolic blood pressure≤90 mmHg;            -   40 bpm≤heart rate≤100 bpm;        -   Normal 12-lead ECG; 120 ms<PR<220 ms, QRS<120 ms, QTc≤430 ms            (for female: QTc≤450 ms);    -   Laboratory parameters within the normal range unless the        Investigator considers an abnormality to be clinically        irrelevant for healthy subjects; however serum creatinine and        hepatic enzymes (AST, ALT) should be strictly below the upper        laboratory norm;    -   Normal metabolic control defined as fasting serum glucose (≤100        mg/dL) and glycosylated hemoglobin (HbA1c≤6.1%);    -   Subjects must be off regular use of prescription drug therapy,        for at least four (4) weeks prior to participation in the study;

Obligations for Female Subjects

-   -   Female subjects of childbearing potential (defined as        pre-menopausal and not surgically sterilized or post-menopausal        for less than 2 years) and sexually active must practice        adequate birth control. Adequate birth control is defined as a        highly effective method of contraception (Pearl index<1%) such        as implants, injectables, combined oral contraceptives or        hormonal IUDs (intrauterine devices). Post-menopausal for the        purposes of this clinical trial include: amenorrhea for 2 or        more years or surgically sterile;    -   Female subjects must have a negative urine beta-human chorionic        gonadotropin (beta-HCG) pregnancy test during the pre-study        screening, and prior to the first clamp;

Regulations

-   -   Having given written informed consent prior to any procedure        related to the study;    -   Covered by Health Insurance System and/or in compliance with the        recommendations of National Law in force relating to biomedical        research;    -   Not under any administrative or legal supervision.        Subjects presenting with any of the following have not been        included in the study:

Medical History and Clinical Status

-   -   Any history or presence of clinically relevant cardiovascular,        pulmonary, gastro-intestinal, hepatic, renal, metabolic,        hematological, neurologic, psychiatric, systemic, ocular or        infectious disease; any acute infectious disease or signs of        acute illness;    -   Presence or history of drug allergy, or allergic disease        diagnosed and treated by a physician;    -   Excessive consumption of beverages with xanthine bases (>4 cups        or glasses/day);    -   Contraindications from (according to normal ranges—if the value        is outside of the normal range the subject can be included if        the Investigator sees this abnormal value as clinically        irrelevant):        -   the medical/surgical history and physical examination        -   laboratory tests (hematology, clinical chemistry, and            urinalysis by dipstick)        -   standard 12-lead electrocardiogram        -   blood pressure and heart rate    -   Any ongoing treatment with prescribed drugs or any regular        treatment with prescribed drugs in the 4 weeks prior to        participation in the study    -   Symptoms of a clinically significant illness in the 3 months        before the study, or of any major internal medical disease in        the 4 weeks before the study which, according to the        Investigator's opinion, could interfere with the purposes of the        study.    -   Presence or sequalae of a disease or other conditions known to        interfere with the absorption, distribution, metabolism, or        excretion of drugs    -   History of drug or alcohol abuse    -   History of hypersensitivity to the study medication or to drugs        with similar chemical structures    -   Progressive fatal disease    -   Pre-planned surgery during the study    -   Blood donation of more than 500 mL during the previous 3 months        No subject has been allowed to enroll in this study more than        once.

General Conditions

-   -   Subject who, in the judgment of the Investigator, is likely to        be non-compliant during the study, or unable to cooperate        because of a language problem or poor mental development or due        to a mental condition rendering the subject unable to understand        the nature, scope and possible consequences of the study    -   Subject in exclusion period of a previous study according to        applicable regulations;    -   Subject is the Investigator or any Sub-Investigator, Research        Assistant, Pharmacist, Study Coordinator, other Staff thereof,        directly involved in the conduct of the protocol;    -   Receipt of an experimental drug within the previous 30 days        before SCR.

Biological Status

-   -   Positive reaction to any of the following tests: HBs antigen,        anti-HCV antibodies, anti-HIV1 antibodies, anti-HIV2 antibodies;    -   Positive results on urine drug screen at SCR        (amphetamines/metamphetamines, barbiturates, benzodiazepines,        cannabinoids, cocaine, opiates);    -   Positive alcohol breath test

Example 3: Treatments Details of Study Treatments

Drug code: HOE901 (Insulin glargine U300 (Lantus ® U100 commercialformulation) formulation) INN: Insulin glargine (recombinant Insulinglargine (recombinant human insulin analogue) human insulin analogue)Formulation: Cartridges for 3 mL solution U300 Cartridges for 3 mLsolution (1 mL contains 10.913 mg 21^(A)-Gly- U100 (1 mL contains 3.637mg 30^(B)a-L- Arg-30^(B)b-L-Arg human 21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L- insulin [equimolar to 300 IU Arg human insulin [equimolarhuman insulin], 90 μg zinc, to 100IU human insulin], 30 μg 2.7 mgm-cresol, 20 mg zinc, 2.7 mg m- cresol, 20 mg glycerol 85%, HCl and NaOHad pH glycerol 85%, HCl and NaOH ad 4.0; specific gravity 1.006 g/mL) pH4.0; specific gravity 1.004 g/mL) Dose/route of administration 0.4 U/kgbody weight; single 0.4 U/kg body weight; single s.c. s.c. injectioninto the injection into the periumbilical periumbilical abdomen after anabdomen after an overnight fast overnight fast Manufacturer:Sanofi-Aventis Manufacturer: Sanofi-Aventis Deutschland GmbHDeutschlandGmbH

Calculation of the Dose for Lantus®/Insulin Glargine Formulation

To calculate the amount of insulin glargine given for each subject (0.4U/kg), the body weight (in kg) has been determined to one decimal placeand the amount of insulin calculated has been rounded up or down tointeger numbers as shown in the following examples: a subject with abody weight of 75.3 kg has received 30 U insulin (75.3×0.4=30.12 whichis rounded down to 30); a subject with a body weight of 74.4 kg hasreceived 30 U insulin (74.4×0.4=29.76, which is rounded up to 30). Thebody weight recorded during Period 1 Day 1 has been used for calculationof study medication dose for Periods 2, 3 and 4, unless the body weightchanged by more than 2 kg compared to Period 1.

The amount in Units has been the same for both insulin glargine U100 andinsulin glargine U300. This specific gravity is the same for both drugproducts. However, given the three times higher concentration of insulinglargine in insulin glargine U300 as compared to insulin glargine U100,the to be injected volume and hence the weight has been ⅓ for insulinglargine U300. The syringes providing the individual dose have beenprepared by weight. The net weight has been documented only in thesource-documentation of the Investigator.

Calculation and Preparation of the Dose for Infusions

TABLE 1 Preparation of infusion Manufac- Dose/Route of Drug Code INNFormulation turer administration Glucose Glucose 20% solution Certified,iv infusion for infusion selected by PROHL Intramed Heparin VialCertified, iv infusion Heparin containing selected by Sodium 5 mLsolution PROHL (5000 IU/mL) 0.9% Sodium Solution Certified, iv infusionSodium Chloride selected by Chloride PROHL

Glucose solution: 20% glucose solution has been infused with theBiostator to keep subjects individual blood glucose at the determinedtarget level. A second infusion pump (part of the Biostator) hasdelivered 0.9% sodium chloride solution to keep the line patent. In casethe amount of 20% glucose solution needed exceeds the infusion capacityof the Biostator, a second glucose infusion pump has been engaged.

Heparin: 10000 IU heparin in 100 mL 0.9% sodium chloride solution havebeen infused into the double lumen catheter at a rate of approximately 2mL/h to keep it patent for blood glucose measurement by the Biostator.

Description of Blinding Methods:

This was a single-blind study. The different volumes of injectionpreclude blinding of the medication. Injection has been done by anauthorized medical person otherwise not involved in the study. TheInvestigator has access to the randomization code.

Method of Assigning Subjects to Treatment Group

The study medication has been administered only to subjects included inthis study following the procedures set out in the clinical studyprotocol.

A randomization schedule has been generated, which has linked therandomization numbers, stratified by gender, to the treatment sequencesof the two Lantus® formulations to be injected at P1 to P4.

In the morning of Day 1 of Period 1, as soon as the Investigator hasconfirmed that subjects fulfill the criteria specified in the protocol,the eligible subjects were randomized by the site. The randomizationnumber was allocated to the subject number subsequently in the order inwhich subjects' eligibility has been confirmed before P1. The firstsubject for a gender stratum qualifying after SCR received the firstrandomization number for the appropriate gender stratum. The nextsubject who qualifies within a stratum received the next randomizationnumber within the stratum.

The randomization number has been used as the treatment kit number toallocate the treatment kit to the subject. Each subject were given thestudy medication carrying the treatment kit number to which he has beenallocated to. The treatment kit containing the IP carried generalinformation, treatment kit number, period number, a field to write thesubject number on the container-box, and additional statements asrequired by local regulations.

Subjects who permanently discontinue from the study retained subjectnumber and randomization number, if already given.

Packaging and Labelling

The study medication has been packed by Sanofi-Aventis Deutschland GmbH,Frankfurt am Main, Germany according to the randomization plan. Thecartridges containing the study medication and the cartons they werepacked in have been labeled with the study number, the randomizationnumber, batch number, storage conditions, Sponsor and the P number.

Supplies of study medication have been received in one shipment. Allcontainers had labels of identical format. Additionally, 1 set of labelsfor syringes has been supplied. Study medication and back-up medicationwere stored in different refrigerators.

Before study medication administration, the Pharmacist or the persondesignated by him has prepared the syringes with the appropriate studymedication and has labeled the syringe with the subject number, therandomization number and the appropriate period according to the studymedication containers.

The content of the labeling was in accordance with the local regulatoryspecifications and requirements.

Storage Conditions

The study medication was stored protected from light at a temperature of+2° C. to +8° C. The study medication was prevented from freezing.During preparation it was not necessary to have the medication protectedfrom light.

Reserve samples (300 cartridges Lantus® U100 and 300 cartridges insulinglargine U 300) were stored in the same secure conditions at the studysite level.

Example 4: Assessment of Investigational Product Activity orPharmacodynamics

Stimulation of insulin receptors by insulin glargine is the mode ofaction. Subsequent peripheral glucose uptake and suppression ofendogenous glucose production comprise the glucodynamic effectsproducing a reduction in blood glucose concentration. The resultingglucose utilization is best characterized by the gauge of glucoserequired to keep the blood glucose concentration constant.

The euglycemic clamp technique has been employed to assess the amount ofglucose needed to keep blood glucose concentrations at 5% below baselinelevel after injection of insulin glargine.

Clinical Assessment Methods

Online blood glucose determination has been done by the Biostator (LifeSciences instruments, Elkhart, Ind., USA) employing the glucose oxidasemethod.

Offsite blood glucose has been determined with a Super GL glucoseanalyzer also using the glucose oxidase method.

Pharmacodynamic Variables/Endpoints

The amount of glucose utilized per unit (dose) of subcutaneouslyinjected insulin is a measure of the glucodynamic effect.

The continuously recorded glucose infusion rate (GIR) is a reflection ofthe time action profile of the injected insulin.

Primary Variable/Endpoint

The primary pharmacodynamic variable is the area under the glucoseinfusion rate time curve within 24 hours [GIR-AUC_(0-24h) (mg·kg⁻¹)].

Secondary Variable/Endpoint

The secondary pharmacodynamic variable is the time to 50%GIR-AUC_(0-24h) [T_(50%)-GIR-AUC_((0-24h)) (h)].

Pharmacokinetics Sampling Times

Blood samples for assessment of serum insulin glargine and C-peptideconcentrations have been taken 1 hour, 30 min and immediately prior tosubcutaneous injection of study medication, thereafter 30 min, 1 hour, 2hours and then bi-hourly up to 24 hours, and 30 hours after injection.

The numbering of insulin glargine samples was P00, P01, P02, P03, P04,etc., the numbering of C-peptide samples was C00, C01, C02, C03, C04,etc (see also study flow chart).

Number of Pharmacokinetic Sampling

A minimum of 18 samples have been taken per clamp visit (P1 to P4). Intotal 72 samples have been taken per subject.

PK Handling Procedure

The exact time of sample collection must be recorded on the CRF. Specialprocedures for storage and shipping of pharmacokinetic samples (insulinglargine, C-peptide) have been used.

Bioanalytical Method

Bioanalysis have been performed using as a basis the Good LaboratoryPractice (GLP) requirements applicable to this type of study identifiedin the OECD Principles of Good Laboratory Practice (as revised in 1997),ENV/MC/CHEM (98)17 and the GLP regulations applicable to the localcountry.

As no back-up samples are available priority is given to determinationof insulin glargine.

Insulin Glargine

Serum insulin glargine concentrations have been determined using aradioimmunoassay (RIA) for human insulin (Insulin RIA kit, ADALTIS,Italy) calibrated for insulin glargine. Kit REF 10624.

The lower limit of quantification (LLOQ) for this assay was 4.92 μU/mL.

C-Peptide

Serum C-peptide concentrations have been determined using aradioimmunoassay (RIA) for C-peptide (C-peptide RIA kit, ADALTIS,Italy). Kit REF C-peptide 10282.

The lower limit of quantification (LLOQ) was 0.090 nmol/L.

Summary of Bioanalytical Method

Analyte insulin, C-peptide Matrix serum Analytical Technique RIA Lowerlimit of 4.92 μU/mL insulin; 0.090 quantification nmol/L C-peptide Assayvolume 100 μL for insulin; 100 μL for C-peptide Method Reference AdaltisS.p.A. Italy; Kit REF 10624 Insulin (Method No. 435VAL02) and Kit REFC-peptide 10282 (Method No. DMPK/FRA/2003-0002)

Pharmacokinetic Variables/Endpoints

The insulin glargine concentration time curve was a measure of thesystemic insulin exposure of subcutanously injected IP.

Primary Variable/Endpoint

The primary pharmacokinetic variable was the area under the seruminsulin glargine concentration time curve [INS-AUC_(0-24h) (μU·h·mL⁻¹)].

Secondary Variable/Endpoint

The secondary pharmacokinetic variable was the time to 50%INS-AUC_(0-24h)

[T_(50%)-INS-AUC_((0-24h)) (h)].

Sampled Blood Volume

Sampled blood volume Archival Blood/Genotyping 0 mL Hematology/Clinical32 mL chemistry/Serology (20 + 12 mL) RBC, Hb, Het (2 × 2 mL) optional 4mL Blood glucose (2 mL/h × 32 × 4) 256 mL Blood glucose (0.3 mL × 4 ×34) 41 mL PK insulin glargine (3.5 mL × 18 × 4) 252 mL Total 585 mL

Measures to Protect Blinding of the Trial

This has been a single-blind study. Bioanalytical determinations havebeen performed after clinical completion. The treatment code has beenknown for reporting of any Serious Adverse Event (SAE) unexpected andreasonably associated with the use of the IP according to either thejudgment of the Investigator and/or the Sponsor.

Example 5: Study Procedures Visit Schedule Screening Procedures

The medical records of each potential subject has been checked beforethe start of the study to determine eligibility for participation. Thesubjects have fasted (except for water) for 10 hours before thescreening examination at SCR.

The following items/examinations have been assessed:

-   -   Age, and race    -   Physical examination (including cardiovascular system, chest and        lungs, thyroid, abdomen, nervous system, skin and mucosae, and        musculoskeletal system)    -   Relevant medical and surgical history (only findings relevant to        the study are to be documented)    -   Anthropometrics: height and weight, calculation of BMI [weight        in kg·(height in m)⁻²]    -   Blood pressure and heart rate (after 5 min in supine and 3 min        upright position)    -   Core body temperature (tympanic)    -   Standard 12-lead ECG    -   Hematology status, clinical chemistry, and urinalysis (by        dipstick)    -   Coagulation status (INR, aPPT)    -   Urine drug screen    -   Alcohol screen (breath analyzer)    -   Normal metabolic control defined as fasting blood glucose (≤100        mg·dL⁻¹) and glycosylated hemoglobin (HbA1c≤6.1%)    -   Hepatitis B/C and HIV test

In case the subject is a screening failure, all data obtained at SCRincluding laboratory results of screening tests were available in thesubject's medical record.

Description by Type of Visit Period(s)

Each study period (P1 to P4) lasted 2 days, Day 1 and Day 2. Day 1 wasthe starting day of the euglycemic clamp and administration of studymedication. Day 2 was day of the end of the euglycemic clamp, whichlasted 30 hours after study medication administration. There was awash-out period of 4-18 days between the study periods (P1-P4). Nostrenuous activity (e.g. mountain biking, heavy gardening etc.) has beenallowed 2 days before each study medication administration. Consumptionof alcoholic beverages, grapefruit juice, and stimulating beveragescontaining xanthine derivatives (tea, chocolate, coffee, Coke™-likedrinks, etc.) and grapefruit has not been permitted from 24 hours beforeuntil completion of the euglycemic clamp. The subjects have fasted(except for water) for 10 hours before Day 1 of each study period (P1 toP4) and remained fasting (except for water) until end of the euglycemicclamp. The subjects had to stay in the clinic for approximately 32 hoursat each clamp visit.

In the morning of Day 1 of Period 1, the 9-digit subject number has beenallocated to the subject, starting with 276001001. The next subject whoqualifies to enter SCR has received the subject number 276001002 etc.The first subject has received the randomization number 101. The nextsubject who qualifies has received the randomization number 102.

Subjects have been asked to ensure that they have had no clinicallysignificant changes in their physical condition and have been compliantwith the general and dietary restrictions as defined in the protocolsince the previous periods. Violation of the study criteria has excludedsubjects from participation in the study. Depending on the kind ofviolation the subject might have been excluded only from the particularperiod, allowing a re-scheduling of the study day. Any protocolviolations have been discussed with the Sponsor on a case-by-case basisin advance.

Any changes in the health condition of the subjects since the lastperiod have been reported in the subject's medical records (source) andthe CRF.

The blood pressure, heart rate and core body temperature (tympanic) havebeen recorded in supine position after at least 5 minutes rest in themorning of Day 1, prior to and after completion of clamp procedures 30hours after each study medication administration (Day 2). Body weight,alcohol screen and RBC, Hb, HcT (only before clamp period of P3 and P4)have been assessed only before starting the clamp in the morning of Day1.

On Day 1 of each period, subjects have been admitted to the clinic at6:30 am. After passing the above described examinations, subjects havebeen prepared with three venous lines. A dorsal hand vein or lateralwrist vein of the left aim has been cannulized in retrograde fashion andconnected to a Biostator (Life Sciences instruments, Elkhart, Ind., USA)in order to continuously draw arterialized venous blood for thedetermination of blood glucose. To achieve arterialization the left handhas been placed in a “Hot-Box” at about 55° C. A second venous line hasbeen placed into the antecubital vein of the left arm and have been usedto collect samples for serum insulin glargine and reference bloodglucose determination. A third vein has been cannulised on thecontralateral forearm allowing the infusion of 20% glucose solution and0.9% saline with the Biostator.

The Biostator determined blood glucose levels and adjusted the glucoseinfusion rate to maintain blood glucose levels at 5% below theindividual fasting blood glucose, determined as the mean of the 3fasting blood glucose values measured 60, 30 and 5 minutes before studymedication administration. Additional blood samples of 0.3 mL for thedetermination of blood glucose have been taken 60, 30, and 5 minutesbefore administration of the study medication to check against alaboratory reference based on the glucose oxidase method.

Approximately at 09:00 am, either insulin glargine U100 (commercialformulation) or insulin glargine U300 have been injected in theperiumbilical area 5 cm lateral to the umbilicus (left, right, left,right) using a standardized skin fold technique. U100 insulin syringes(manufacturer: Beckton & Dickinson) of 0.5 mL volume with a needle of0.30 mm×8 mm (30G) have been used.

The study medication was labeled with their respective treatment kitnumber, subject number (to be documented on the container-box afterrandomization), and Period number (see Section 8.5 Packaging andLabeling).

After study medication administration, infusion of 20% glucose solutionhave commenced at a variable rate once blood glucose level has fallen by5% from the individual fasting level to maintain that level. Theduration of the clamp period have been 30 hours. The rate of glucosedelivery have been adjusted by the Biostator in response to changes inblood glucose at 1 minute intervals using a predefined algorithm. Theblood glucose values from the Biostator have been checked against alaboratory reference based on the glucose oxidase method at 30 minutesintervals for the entire clamp. If necessary the Biostator have beenre-calibrated according to results of the laboratory reference method.Subjects remained in supine position during the period of clamping.

Blood samples for determination of serum insulin glargine and C-peptideconcentrations have been taken 1 hour, 30 min and immediately beforemedication and thereafter 30 min, 1 hour, 2 hours and then bi-hourly upto 24 hours, and 30 hours after administration of study medication.

On day 2 of each study period (P1 to P4), a meal have been served afterthe euglycemic clamp has been completed. Blood pressure, heart rate, andcore body temperature (tympanic) have been recorded, and a sample forblood glucose has been taken. The subjects have been discharged from theclinic after their safety has been ensured by the Investigator.

Injection sites have been observed during the entire clamp period. Anychanges in the health condition of the subjects have been reported inthe subject's medical records (source) and the CRF.

Safety Hematology

RBC, Hb and Hct at P 3 have been analyzed for incurring anemia at P 4.If positive, the interval between P 3 and P 4 have been extended to themaximum allowed 18 days and an additional RBC, Hb and Hct assessmentmade prior to P 4.

Discharge Procedures

Subjects have returned for an EOS visit between 4 to 14 days after P4.Subjects have fasted (apart from water) for 10 hours. Any changes in thehealth condition of the subjects since the last period have beenreported in the subject's medical records (source) and the CRF.

The following items/examinations have been assessed:

-   -   Physical examination (including cardiovascular system, chest and        lungs, thyroid, abdomen, nervous system, skin and mucosae, and        musculoskeletal system)    -   Weight    -   Blood pressure and heart rate (after 5 min in supine position)    -   Core body temperature (tympanic)    -   Standard 12-lead ECG    -   Hematology status, clinical chemistry, and urinalysis (by        dipstick)    -   β-HCG test in urine (only for females)

The subjects have been discharged on Day 2 of each period, after acomplete review by the Investigator of the available safety data.

Collection Schedule for Biological Samples Blood SCR (Screening):

-   -   Hematology, Clinical Chemistry, HbA1c, Serology (Hepatitis B/C        test, HW test): approximately 20 mL of blood have been        collected.

P1 to P4 (Day 1 and 2):

-   -   Blood glucose        -   Biostator has automatically measured blood glucose at one            minute intervals for the entire clamp period, including the            period prior to study medication. The volume of blood needed            by the Biostator have been 2 mL·h⁻¹. An estimated 252 mL            blood volume have been needed for glucose readings with the            Biostator for the four periods. Blood samples (0.3 mL) for            checking blood glucose values from Biostator have been            collected 60, 30, 5 and 0 minutes prior to dosing and at 30            minute intervals after dosing until end of the clamp (30            hours). An estimated 41 mL blood volume have been collected            for the four periods.    -   Serum insulin glargine and C-peptide concentrations        -   Venous blood samples (3.5 mL) have been collected 1 hour, 30            min and immediately prior to dosing, 30 min, 1 hour, 2 hours            and then bi-hourly up to 24 hours, and 30 hours after            dosing. An estimated 252 mL blood volume have been collected            for the four periods. Determination of insulin glargine has            been given priority. Spare samples only have been used for            determination of C-peptide concentration.    -   RBC, Hb, Hct        -   Venous blood have been collected before commencing clamp            period 3 and 4.        -   Approximately 4 mL of blood have been collected for the two            periods.

End-of-Study (EOS) Visit:

-   -   Hematology, Clinical Chemistry: approximately 12 mL of blood        have been collected.    -   β-HCG test in urine (only for females)

Total Blood Volume SCR-EOS:

In total, approximately 585 mL blood have been collected for eachsubject during the entire study.

Urine

Qualitative urine drug screen have been conducted at SCR and EOS. Urinedrug screen consists of amphetamines/metamphetamines, barbiturates,benzodiazepines, cannabinoids, cocaine, opiates. Qualitative safetyurinalysis with dipsticks have been conducted at SCR and EOS. Safetyurinalysis consists of analysis for: pH, protein, glucose, blood,erythrocytes, leukocytes, bilirubin, urobilinogen, ketone, specificgravity, and nitrite.

Measurement Schedule for Other Study Variables

Physical examination have been performed at SCR and EOS.

Core body temperature (tympanic) have been taken at SCR, P1 to P4 beforeand after the clamp period, and at EOS.

Blood pressure and heart rate have been measured after about 5 minutesrest in a supine position, and also after 3 minutes in an uprightposition at SCR and EOS. In P1 to P4 blood pressure and heart rate havebeen recorded in supine position after at least 5 minutes prior to startof clamp procedures in the morning of day 1, and after completion ofclamp procedures 30 hours after each study medication administration(day 2).

Electrocardiograms (standard 12-lead) have been recorded at SCR and EOS.

Body weight and height have been measured at SCR. The body weight havebeen recorded in the morning of Day 1 of P1 to P4 (prior toadministration of study medication) and at EOS. Alcohol screen (ethanol,breath analyzer) have been conducted at SCR and EOS, and in the morningof Day 1 of P1 to P4 (prior to administration of study medication).

Study Restriction(s)

From Day-1 evening (P1 to P4) and throughout the Periods (clamp days),the subjects have refrained from drinking alcohol, tea, coffee, citrusor cola beverages, smoking. Eating citrus fruits was also prohibitedthroughout the study. The subjects have been requested to follow astable lifestyle throughout the duration of the trial, until the lastcontrol, with no intensive physical activity.

Definition of Source Data

All evaluations listed below that are reported in the CRF were supportedby appropriately signed identified source documentation related to:

-   -   subject identification, medical history;    -   clinical examination, vital signs, body weight and height;    -   laboratory assessments, ECG;    -   pharmacokinetic time points;    -   dates and times of visits and assessments;    -   administration dates and times, and site of injection;    -   AEs;    -   duration of clamp (start and end times)    -   Other

The CRF have been considered as source documentation for other items.

Example 6: Statistical Considerations

This example provides information for the statistical analysis plan forthe study. A statistical analysis plan have been drafted prior toinclusion of subjects.

Determination of Sample Size

INS-AUC_((0-24h)) have been the primary parameter for which thereforethe sample size calculation was performed.

For the purpose of this sample size calculation, several within-subjectSD_(within) of natural log-transformed INS-AUC_((0-24h)) between 0.125and 0.225 were considered. A sample size calculation method for anaverage bioequivalence approach was used for a 4-period, 2-treatment,2-sequence cross-over design. If the 90% CIs for the formulation ratiohave been wholly contained within [0.80-1.25], then averagebioequivalence have been concluded for the parameter.

Study HOE901/1022 was the basis for assumptions on variability. Based onthe statistical analysis of study HOE901/1022, a value of 0.175 could beexpected for the within subject standard deviation (SD_(within)) on thenatural log-transformed scale.

The table below indicates the number of subjects required to demonstrateaverage bioequivalence of the ratio of adjusted geometric means (testversus reference formulation) using the bioequivalence referenceinterval: [0.80-1.25], assuming a true ratio between 0.85 and 1.15 with90% power.

TABLE 2 Required total number of subjects to achieve a power of at least90% SD(within) on natural log-scale Assumed 0.125 0.15 0.175 0.2 0.225true ratio N N N N N 0.85 38 54 72 94 120 0.90 12 16 20 26 32 0.95 6 810 14 16 1.00 6 6 8 10 12 1.05 6 8 10 12 16 1.10 10 14 18 22 28 1.15 2030 40 50 64 N = total number of subjects

With this design, 20 subjects (10 per sequence) are required todemonstrate equivalence of the two insulin glargine formulations, with90% power, allowing true ratio of 0.9, if the true SD_(within) onnatural log scale is 0.175.

A number of 24 randomized subjects accounts for potential cases ofwithdrawals.

Subject Description Disposition of Subjects

A detailed summary of subject accountability including count of subjectsincluded, randomized, exposed (i.e. received any amount of studymedication), completed (i.e. subjects who completed all study treatmentperiods), discontinued along with the main reasons for discontinuationhave been generated for each sequence and for all subjects in total.

Subject disposition at the final visit have been presented in a listingincluding sequence group, disposition status at the end of the studywith the date of last administration of study drug, date of final visit,reason for discontinuation. All withdrawals from the study, taking placeon or after the start of the first study drug administration, have beenfully documented in the body of the clinical study report (CSR).

Protocol Deviations

Prior to data base lock, the compliance with the protocol have beenexamined with regard to inclusion and exclusion criteria, treatmentcompliance, prohibited therapies, and timing and availability of plannedassessments. Protocol deviations have been identified by the study teambefore database lock and listed in the Data Review Report, includingmissing data and IP discontinuations, and classified as minor or majordeviations.

Individual deviations to inclusion and exclusion criteria as reported bythe Investigator have been listed.

Other deviations have been listed by and/or described in the body of theCSR.

Analysis Population Population to be analyzed

Subjects excluded from any analysis population have been listed withtreatment sequence, and with reason for exclusion. Any relevantinformation have been fully documented in the CSR.

In the event of subjects having received treatments that differed fromthose assigned according to the randomization schedule, analyses havebeen conducted according to the treatment received rather than accordingto the randomized treatment.

Pharmacokinetic Population

All subjects without any major deviations related to study drugadministration, and for whom PK parameters are available, have beenincluded in the pharmacokinetic population. For subjects withinsufficient PK profiles in some but not all study days, parameters ofthe sufficient profiles have been included in the analysis.

Pharmacodynamic Population

All subjects without any major deviations related to study drugadministration, and for whom PD parameters are available, have beenincluded in the pharmacodynamic population. For subjects withinsufficient GIR-profiles in some but not all study days, parameters ofthe sufficient profiles have been included in the analysis.

Safety Population

Safety evaluation have been based on subjects who received a dose ofstudy drug (exposed population), regardless of the amount of treatmentadministered, including subjects prematurely withdrawn.

Demographic and Baseline Characteristics

Subject Demographic Characteristics, Medical History and Diagnoses

The following data have been collected: sex, age at screening, height,weight, and race. Body mass index (BMI) per subject have been calculatedfrom body weight and height data:

BMI=body weight [kg]·(height [m])⁻²

All variables concerning demographic and background characteristics havebeen listed individually and summarized.

Deviations from inclusion criteria related to medical history anddiagnoses have been listed and described individually.

Baseline Pharmacodynamic Parameters

Baseline blood glucose levels have been summarized by sequence.

Baseline Safety Parameters

For safety variables, the latest scheduled value before study drugadministration within the period or within the study, whatever isapplicable for the variable, have been taken as the baseline value. Ifthe baseline pre-dosing value is rechecked before dosing, the recheckedvalue have been considered as the baseline and used in statistics.

Extent of Study Treatment Exposure and Compliance

Details of study drug dosing and complementary information have beenlisted individually and summarized if appropriate.

Prior/Concomitant Medication/Therapy

Prior and concomitant medications/therapies (if any) have been codedaccording to the World Health Organization-Drug Reference List (WHO-DRL)and have been listed individually.

Analysis of Pharmacodynamic Variables Description of PharmacodynamicVariable(s)

In order to achieve comparability between the subjects under the bodyweight depending insulin dosing, all values for GIR have been divided bythe subject's body weight in kg for analysis. Thus, GIR in the belowalways refers to the body weight standardized glucose infusion rate.

Primary PD Variable has been:

-   -   Area under the body weight standardized glucose infusion rate        time curve

[GIR-AUC_((0-24h)) (mg·kg⁻¹)] Secondary PD variable has been:

-   -   Time (h) to 50% of GIR-AUC_((0-24h)) [T_(50%)-GIR-AUC_((0-24h))        (h)]

The following additional PD variables have been derived:

-   -   Area under the body weight standardized glucose infusion rate        time curve up to end of clamp [GIR-AUC_((0-end)) (mg·kg⁻¹)]    -   Fractional areas under the body weight standardized glucose        infusion rate time curve [GIR-AUC_((4-20h)), GIR-AUC_((0-12h)),        GIR-AUC_((12-24h)) (mg·kg⁻¹)]    -   Maximum body weight standardized glucose infusion rate        [GIR_(max) (mg·kg⁻¹·min⁻¹)]    -   Time to GIR_(max) [GIR-t_(max) (h)]

In order to provide meaningful and reliable data, the value forGIR_(max) and correspondingly the time to GIR_(max) have been derivedfrom a smoothed GIR curve for each subject.

Primary Analysis

To estimate relative bioefficacy (activity) for GIR-AUC_((0-24h))(mg·kg⁻¹), the untransformed parameter has been analyzed with a linearmixed effects model.

The mixed model includes fixed terms for sequence, period, formulation,and random terms for subject within sequence, with formulation specificbetween-subject and within-subject variances and subject-by-formulationvariance. Point estimate and 90% confidence interval for the formulationratio (T/R) have then been obtained based on Fieller's theorem [Fieller,1954].

Equivalent bioefficacy (activity) has been concluded if the confidenceinterval for the formulation ratio has been placed within [0.80-1.25].

Assumptions for the distribution of the variable has been checked.

Secondary Analysis/Analysis of Secondary Variables

Individual and mean body weight standardized GIR-profiles as well asmean percentage cumulative profiles over time have been plotted.

PD parameters have been listed individually, and descriptive statisticshas been generated.

Formulation ratios (T/R) with confidence limits have been derived forfractional GIR-AUCs (mg·kg⁻¹) and maximum standardized glucose infusionrate [GIR_(max) (mg·kg⁻¹·min⁻¹)] using the corresponding linear mixedeffects model as described for the primary analysis.

Time to 50%-GIR-AUC (h) and time to GIR_(max) [GIR-t_(max) (h)] havebeen analyzed non-parametrically.

Performance of Clamp

Individual profiles of blood glucose concentration have been plotted.

Analysis of Safety Data

All summaries of safety data have been based on the safety population.

The individual on-treatment phase for analysis of safety data havestarted with the first administration of study medication and has endedwith the EOS visit.

Adverse Events

All AEs have been coded using MedDRA (version in use).

Definitions

Treatment Emergent AEs

All AEs have been classified as follows:

-   -   Treatment-emergent AEs (TEAEs): AEs that occurred during the        on-treatment period for the first time or worsened during the        on-treatment period, if present before;    -   Non-treatment-emergent AEs (NTEAEs): AEs that occurred outside        the on-treatment period without worsening during the        on-treatment period;

Assignment to Formulations

For analysis purposes, each TEAE has been assigned to the lastformulation given before onset and/or worsening of the AE. If a TEAEdevelops on one formulation and worsens under a later formulation, ithas been considered a TEAE for both formulations.

Missing Information

In case of missing or inconsistent information, an AE has been countedas a TEAE, unless it can clearly be ruled out that it is not a TEAE (e.g. by partial dates or other information).

If the start date of an AE is incomplete or missing, it has been assumedto have occurred after the first administration of study medicationexcept if an incomplete date indicated that the AE started prior totreatment.

Treatment-Emergent Adverse Events

All AEs have been listed individually. They have been summarized byformulation, including summary by system organ class.

Deaths, Serious and Other Significant Adverse Events

If any such cases, deaths, serious AEs, and other significant AEs havebeen listed individually and described in the study report in detail.

Adverse Events leading to Treatment Discontinuation AEs leading totreatment discontinuation have been listed individually and described inthe study report in detail.

Clinical Laboratory Evaluations

Potentially clinically significant abnormalities (PCSA) and out-of-rangecriteria have been defined in the statistical analysis plan of thisstudy. Definitions of potentially clinically significant abnormalities(PCSA) and out-of-range definitions have been reported by parameter.

Individual data have been listed by subject and by visit, as well ascomplementary information.

Subjects with values out of normal ranges and subjects with PCSAs havebeen analyzed by formulation, and overall for end of study evaluation.Subjects with post-baseline PCSAs have been listed.

Vital Signs

Potentially clinically significant abnormalities (PCSA) and out-of-rangecriteria have been defined in the statistical analysis plan of thisstudy. Definitions of PCSA and out-of-range definitions have beenreported by parameter.

Subjects with PCSAs have been analyzed by formulation, and overall forend of study evaluation. Subjects with post-baseline PCSAs have beenlisted.

Raw values and derived parameters have been summarized by formulation,and overall for end of study evaluation. Individual data have beenlisted by subject and by visit with flags for abnormalities, as well ascomplementary information.

ECG

Potentially clinically significant abnormalities (PCSA) and out-of-rangecriteria have been defined in the statistical analysis plan of thisstudy. Definitions of PCSA and out-of-range definitions have beenreported by parameter.

Subjects with PCSAs at end of study have been analyzed overall. Subjectswith post-baseline PCSAs have been listed.

Raw values and derived parameters at SCR and at EOS have been summarizedoverall. Individual data have been listed by subject and by visit withflags for abnormalities, as well as complementary information.

Analysis of Pharmacokinetic Data Pharmacokinetic Parameters

Actual relative times have been used to derive PK parameters.

Primary variable has been

-   -   INS-AUC_((0-24h)) (μU·h·mL⁻¹)

Secondary PK variable has been

-   -   Time (h) to 50% of INS-AUC_((0-24h))) [T_(50%)-INS-AUC_((0-24h))        (h)]

The following additional PK variables have been derived:

-   -   Fractional INS-AUCs [INS-AUC_((4-20h)), INS-AUC_((0-12h)),        INS-AUC_((12-24h)) (μU·h·mL⁻¹)]    -   INS-AUC up to end of clamp [INS-AUC_((0-end)) (μU·h·mL⁻¹)]    -   Maximum serum insulin concentration [INS-C_(max) (μU·mL⁻¹)]    -   Time to INS-C_(max) [INS-T_(max) (h)]

Statistical Analysis Descriptive Analyses

Descriptive statistics of concentration data have been presented byprotocol times.

Individual and mean serum insulin concentration profiles have beenplotted.

Serum insulin concentrations have been individually listed anddescriptive statistics per time point have been generated.

Descriptive statistics of PK parameters have been generated byformulation.

Profiles of C-peptide have been plotted and characterized descriptively.

Primary Analysis

To estimate relative bioavailability for INS-AUC_((0-24h)), thelog-transformed parameter has been analyzed with a linear mixed effectsmodel.

The mixed model included fixed terms for sequence, period, formulation,and random terms for subject within sequence, with formulation specificbetween-subject and within-subject variances and subject-by-formulationvariance.

For INS-AUC_((0-24h))), point estimate and 90% confidence intervals forthe formulation ratio (T/R) have been obtained by computing estimatesand 90% confidence intervals for the difference between formulationmeans within the mixed effects model framework, and then converting tothe ratio scale by the antilog transformation.

Equivalent bioavailability has been concluded if the confidence intervalfor the formulation ratio has been placed within [0.80-1.25].

Analyses of Secondary and Additional PK Parameters

Time to 50%-INS-AUC (h) and time to maximum concentration [INS-T_(max)(h)] have been analyzed non-parametrically.

Log-transformed fractional INS-AUCs and INS-AUC_((0-end)) (μU·h·mL⁻¹)and maximum serum insulin glargine concentration [INS-C_(max) (μU·mL⁻¹)]have been analyzed with the corresponding linear mixed effects model asdescribed for the primary analysis. Point estimators and confidenceintervals have been reported.

C-Peptide

As available, profiles of C-peptide have been plotted and characterizeddescriptively.

PK/PD Analysis

PK/PD analyses have been performed in an explorative manner, ifappropriate.

Example 6: Study Results Subject Disposition

A total of 35 subjects, 11 women and 24 men, were screened of which 24healthy eligible subjects were enrolled, randomized and received atleast one dose of study medication. Of the 24 randomized subjects, 1subject withdrew from the study on own request after the first dosetreatment period. Twenty-three (23) subjects completed the studyaccording to the protocol and were included in the pharmacodynamic (PD)and pharmacokinetic (PK) analyses. All 24 treated subjects were includedin the safety evaluation.

There were no major protocol deviations.

Demographics Characteristics

The following data were collected: sex, age at screening, height,weight, and race. Body mass indexes (BMI) per subject were calculatedfrom body weight and height data: BMI=body weight [kg]·(height [m])⁻².

TABLE 3 Summary of Subject Characteristics - Safety Population SexStatistics/ Male Female All Category (N = 17) (N = 7) (N = 24) Age N 177 24 (years) Mean (SD) 34.8 (6.4) 39.1 (5.6) 36.1 (6.3) (Min, Max) (25,45) (32, 45) (25, 45) Weight N 17 7 24 (kg) Mean (SD) 80.25 (10.42)64.17 (5.70) 75.56 (11.82) (Min, Max)  (65.9, 101.2) (57.6, 74.2) (57.6, 101.2) Height N 17 7 24 (cm) Mean (SD) 180.6 (6.0) 166.3 (5.1)176.4 (8.7) (Min, Max) (171, 189) (158, 174) (158, 189) BMI N 17 7 24(kg/m2) Mean (SD) 24.55 (2.40) 23.19 (1.55) 24.15 (2.24) (Min, Max)(20.5, 28.3) (21.4, 24.6) (20.5, 28.3) Race Black 1 (5.9) 0 (0) 1 (4.2)[n (%)] Caucasian/ 16 (94.1) 7 (100) 23 (95.8) white

Clamp Performance

The two treatment groups, Lantus U 100 and Lantus U 300, were similarregarding the individuals' fasting baseline blood glucoseconcentrations, which served to define the individuals' glucose clamplevel. The duration of the clamps after dosing was 30 hours and the samein all treatment periods.

Primary Endpoints

Equivalence in bio-availability (exposure) for Lantus U 100 and Lantus U300 was not established. Equivalence in bio-efficacy (activity) forLantus U 100 and Lantus U 300 was not established.

Primary Variables

The area under the serum insulin glargine concentration time curve from0 to 24 hours (INS-AUC_((0-24h))) was not equivalent for Lantus U 100and Lantus U 300. The exposure was less by about 40% with U300. The areaunder the GIR versus time curve from 0 to 24 hours (GIR-AUC_((0-24h)))was not equivalent for Lantus U 100 and Lantus U 300. The activity wasless by about 40% with U300.

Secondary Variables

The time to 50% of INS-AUC_((0-24h)) (h) was similar for Lantus U 100and Lantus U 300. The time to 50% of GIR-AUC_((0-24h)) (h) was greaterby 0.545 (h) (0.158-1.030) for Lantus U 300, which was statisticallysignificant.

Safety

No serious adverse events (AEs) were reported. Five (5) subjects pertreatment (test and reference) reported a total 14 TEAEs, all of whichwere of mild to moderate intensity, and resolved without sequalae. Themost frequently reported event was headache (4 subjects per treatment)followed by nausea, vomiting and pyrexia (1 subject each on U 100), andprocedural pain (1 subject on U 300). Of note, headache is a commonobservation for clamp studies and is related to the infusion ofhyper-osmolaric glucose solutions. However, a link to theinvestigational products cannot be excluded. No injection site reactionswere reported.

Conclusions

Insulin glargine U 100 and insulin glargine U 300 are not equivalent inbio-availability (exposure) and bio-efficacy (activity). Exposure andactivity after insulin glargine U300 were less by about 40% as comparedto exposure and activity after administration of the same amount (0.4U/kg) from insulin glargine U100.

Insulin glargine U300 did, however, show an even flatter PK (exposure)and PD (activity) profile than insulin glargine U100, as would bedesired for a basal insulin. These surprising and unexpected differencesin exposure and activity between insulin glargine U100 and insulinglargine U300 formulations after the same s.c. dose to healthy subjectsare effectively shown in the figures below. Of note, at the same timeblood glucose was constant.

Administration of insulin glargine U 300 was without safety andtolerability issues.

Example 7: Study Rationale for Study Comparing the Glucodynamic Activityand Exposure of Three Different Subcutaneous Doses of Insulin GlargineU300

Results from the study in healthy subjects (see examples 1-6) showed theinequivalence in exposure and effectiveness between Lantus® U100 andinsulin glargine U300. Subjects received the same dose of insulinglargine (0.4 U/kg) for U100 and U300, but delivery of the sameunit-amount from U300 produced about 40% less exposure and effect thandelivery from U100. Insulin glargine U300 did, however, show an evenflatter pharmacodynamic profile than Lantus® U100, as would be desiredfor a basal insulin.

A new study described in the following examples therefore compares theglucodynamic activity and exposure of three different subcutaneous dosesof insulin glargine U300 versus a standard dose of Lantus® U100 ascomparator in a euglycemic clamp setting with type 1 diabetes patients.This study aims to approximate an U300 dose that is equieffective to 0.4U/kg Lantus® U100 as assessed by parameters of blood glucose disposalprovided by the clamp technique.

Insulin glargine exposure is assessed from concentration-time profilesafter subcutaneous administration and activity as glucose utilizationper unit insulin.

The study is designed to assess the metabolic effect and exposure ofdifferent insulin glargine U300 doses compared to a standard dose ofLantus® U100 in a euglycemic clamp setting in subjects with diabetesmellitus type 1. The study comprises 4 treatments (R, T₁, T₂ and T₃), 4treatment periods (TP1-4) and 4 sequences. There is one screening visit(D-28 to D-3), 4 treatment visits (D1 to D2 in TP1 to TP4), and oneend-of-study visit (between D5 to D14 in after last dosing) with finalassessment of safety parameters.

Subjects are exposed to each treatment R, T₁, T₂ and T₃ once in across-over, double-blind and randomized manner according to a Latinsquare design. This design is considered appropriate to evaluate thepharmacological effect and exposure of different insulin glargine U300doses compared to Lantus® U100.

The Lantus® U100 dose of 0.4 U/kg selected for the study is wellcharacterized to provide euglycemia in type 1 diabetes patients and hasbeen readily investigated in other clamp studies with type 1 diabetespatients.

Three different doses are tested for insulin glargine U300, 0.4, 0.6 and0.9 U/kg. This dose range allows intrapolating an approximate doseequieffective to 0.4 U/kg Lantus® U100. The dose of 0.4 U/kg of insulinglargine U300 has already been tested in healthy volunteers (seeexamples 1-6) and was found to be less active than 0.4 U/kg Lantus® U100within 30 hours, the predefined end of observation period. Bioactivityof 0.4 U/kg insulin glargine U300 as measured by the total glucosedisposition was 39.4% lower than that of reference medication (0.4 U/kgLantus® U100). A correspondingly higher dose of insulin glargine U300,e.g. 0.6 U/kg insulin glargine U300, was expected to result in anapproximately equivalent glucodynamic activity compared to 0.4 U/kgLantus® U100. Moreover, the proportional dose escalation allowsexploring exposure and effect profiles for dose-proportionality.

A study in patients with type 1 diabetes avoids confounding impact ofendogenous insulin and better permits assessment of exposure andduration of action. Furthermore, the lack of an assay specific forinsulin glargine forces to use an assay which reads all endogenousinsulin. Thus, any added source of insulin other than exogenous insulinglargine would cause falsely too high insulin concentrations.

This study has a cross over design; for practical and ethical reasonsnot more than 3 U300 doses will be compared to Lantus® U100. Assessmentof glucodynamic activity of long acting insulin products requires aeuglycemic clamp setting for up to 36 hours owed to the extendedduration of action.

The active pharmaceutical ingredient, insulin glargine, is the same inboth formulations, U100 and U300. The doses used in this study arewithin the range of regular use. Although an overall risk ofhypoglycemia is not completely excluded, it is controlled by theeuglycemic clamp technique.

Pharmacodynamics

The pharmacodynamic activity of insulin glargine is evaluated by theeuglycemic clamp technique in type 1 diabetes patients, which is theestablished standard procedure to evaluate the effect of exogenousadministered insulin products on blood glucose disposal.

Parameters specific for assessment of glucose disposition in aeuglycemic clamp setting are the body weight standardized glucoseinfusion rate (GIR), total glucose disposed, GIR-AUC₀₋₃₆, and times to agiven percentage of GIR-AUC₀₋₃₆ such as time to 50% of GIR-AUC₀₋₃₆.

Ancillary parameters are the maximum smoothed body weight standardizedGIR, GIR_(max), and Time to GIR_(max), GIR-T_(max).

Duration of action of insulin glargine is derived from the time betweendosing and pre-specified deviations above the euglycemic (clamp) level.

Glucose monitoring is performed for 36 hours due to the long duration ofaction of insulin glargine after subcutaneous administration

Pharmacokinetics

Due to the sustained release nature of insulin glargine there is a lackof pronounced peaks in the concentration profile. Therefore, the time to50% of INS-AUC (T_(50%) INS-AUC₀₋₃₆) is calculated as a measure for thetime location of the insulin glargine exposure profile, and INS-C_(max)and INS-T_(max) will serve as additional measures.

Primary Study Objectives

The primary objective of the study is to assess the metabolic effectratios of three different insulin glargine U300 doses versus 0.4 U/kgLantus® U100.

Secondary Study Objectives

The secondary objectives of the study are to assess the exposure ratiosof three different insulin glargine U300 doses versus 0.4 U/kg Lantus®U100, to compare the duration of action of different insulin glargineU300 doses versus 0.4 U/kg Lantus® U100, to explore the dose responseand dose exposure relationship of insulin glargine U300, and to assesthe safety and tolerability of insulin glargine U300 in subjects withtype 1 diabetes.

Example 8: Study Design, Description of the Protocol

Phase I, single-center, double-blind, randomized, cross-over (4treatments, 4 treatment periods and 4 sequences; Latin square), activecontrol, with a wash-out duration between treatment periods (5-18 days,preferred 7 days) in male and female subjects with type 1 diabetesmellitus receiving single-doses of insulin glargine at

-   -   0.4 U/kg Lantus® U100 (=Reference R)    -   0.4 U/kg Insulin glargine U300 (=Test T₁)    -   0.6 U/kg Insulin glargine U300 (=Test T₂)    -   0.9 U/kg Insulin glargine U300 (=Test T₃)

The four treatments R and T₁₋₃ are given cross-over in four treatmentperiods (TP 1 to TP 4) with the four-sequences

-   -   R-T₁-T₂-T₃    -   T₃-R-T₁-T₂    -   T₂-T₃-R-T₁    -   T₁-T₂-T₃-R        randomly assigned to the subjects (1:1:1:1 ratio).

Duration of Study Participation

-   -   Total study duration for one subject: about 4-11 weeks (min-max        duration, depending on wash-out period, excl. screening)    -   Duration of each part of the study for one subject:        -   Screening: 3 to 28 days (D-28 to D-3)        -   Treatment Period 1-4: 2 days (1 overnight stay)        -   Washout: 5-18 days (preferentially 7 days between            consecutive dosings)        -   End-of-study visit: 1 day between D5 and D14 after last            study drug administration

Example 9: Selection of Subjects

Number of subjects planned: At least 24 subjects are to be enrolled tohave 20 evaluable subjects.

Inclusion Criteria Demography

-   I 01. Male or female subjects, between 18 and 65 years of age,    inclusive, with diabetes mellitus type 1 for more than one year, as    defined by the American Diabetes Association (American Diabetic    Association. Report of the Expert Committee on the Diagnosis and    Classification of Diabetes Mellitus. Diabetes Care 1998; 21:5-19)-   I 02. Total insulin dose of <1.2 U/kg/day-   I 03. Body weight between 50.0 kg and 95.0 kg inclusive if male,    between 50.0 kg and 85.0 kg inclusive if female, Body Mass Index    between 18.0 and 30.0 kg/m² inclusive

Health Status

-   I 04. Fasting negative serum C-peptide (≤0.3 nmol/L)-   I 05. Glycohemoglobin (HbA1c)≤9.0%-   I 06. Stable insulin regimen for at least 2 months prior to study    (with respect to safety of the subject and scientific integrity of    the study)-   I 07. Normal findings in medical history and physical examination    (cardiovascular system, chest and lungs, thyroid, abdomen, nervous    system, skin and mucosae, and musculo-skeletal system), unless the    investigator considers any abnormality to be clinically irrelevant    and not interfering with the conduct of the study (with respect to    safety of the subject and scientific integrity of the study)-   I 08. Normal vital signs after 10 minutes resting in the supine    position: 95 mmHg<systolic blood pressure<140 mmHg; 45    mmHg<diastolic blood pressure<90 mmHg; 40 bpm<heart rate<100 bpm-   I 09. Normal standard 12-lead ECG after 10 minutes resting in the    supine position; 120 ms<PQ<220 ms, QRS<120 ms, QTc≤440 ms if male,    ≤450 ms if female-   I 10. Laboratory parameters within the normal range (or defined    screening threshold for the Investigator site), unless the    Investigator considers an abnormality to be clinically irrelevant    for diabetes patients; however serum creatinine should be strictly    below the upper laboratory norm; hepatic enzymes (AST, ALT) and    bilirubin (unless the subject has documented Gilbert syndrome)    should be not above 1.5 ULN

Female Subjects Only

-   I 11. Women of childbearing potential (less than two years    post-menopausal or not surgically sterile for more than 3 months),    must have a negative serum β-HCG pregnancy test at screening and a    negative urine β-HCG pregnancy test at Day 1 on TP1 to TP4 and must    use a highly effective method of birth control, which is defined as    those which result in a low failure rate (i.e. less than 1% per    year) according to the Note for guidance on non-clinical safety    studies for the conduct of human clinical trials for pharmaceuticals    (CPMP/ICH/286/95, modifications). During the entire study female    subjects of child bearing potential must use two independent methods    of contraception, e.g. diaphragm and spermicide-coated condom. The    use of a condom and spermicidal creams is not sufficiently reliable.    -   For postmenopausal women with presence of less than two years        post-menopausal, and not surgically sterile for more than 3        months, the hormonal status will be determined (FSH>30 IU/L,        estradiol<20 pg/mL)

Exclusion Criteria Medical History and Clinical Status

-   E 01. Any history or presence of clinically relevant cardiovascular,    pulmonary, gastro-intestinal, hepatic, renal, metabolic (apart from    diabetes mellitus type 1), hematological, neurological, psychiatric,    systemic (affecting the body as a whole), ocular, gynecologic (if    female), or infectious disease; any acute infectious disease or    signs of acute illness-   E 02. More than one episode of severe hypoglycemia with seizure,    coma or requiring assistance of another person during the past 6    months-   E 03. Frequent severe headaches and/or migraine, recurrent nausea    and/or vomiting (more than twice a month)-   E 04. Blood loss (>300 ml) within 3 months before inclusion-   E 05. Symptomatic hypotension (whatever the decrease in blood    pressure), or asymptomatic postural hypotension defined by a    decrease in SBP equal to or greater than 20 mmHg within three    minutes when changing from the supine to the standing position-   E 06. Presence or history of a drug allergy or clinically    significant allergic disease according to the Investigator's    judgment-   E 07. Likelihood of requiring treatment during the study period with    drugs not permitted by the clinical study protocol-   E 08. Participation in a trial with any investigational drug during    the past three months-   E 09. Symptoms of a clinically significant illness in the 3 months    before the study, which, according to the investigator's opinion,    could interfere with the purposes of the study-   E 10. Presence of drug or alcohol abuse (alcohol consumption>40    grams/day)-   E 11. Smoking more than 5 cigarettes or equivalent per day, unable    to refrain from smoking during the study-   E 12. Excessive consumption of beverages with xanthine bases (>4    cups or glasses/day)-   E 13. If female, pregnancy (defined as positive β-HCG test),    breast-feeding Interfering substance-   E 14. Any medication (including St John's Wort) within 14 days    before inclusion, or within 5 times the elimination half-life or    pharmacodynamic half-life of that drug, whichever the longest and    regular use of any medication other than insulins in the last month    before study start with the exception of thyroid hormones,    lipid-lowering and antihypertensive drugs, and, if female, with the    exception of hormonal contraception or menopausal hormone    replacement therapy; any vaccination within the last 28 days

General Conditions

-   E 15. Subject who, in the judgment of the Investigator, is likely to    be non-compliant during the study, or unable to cooperate because of    a language problem or poor mental development-   E 16. Subject in exclusion period of a previous study according to    applicable regulations-   E 17. Subject who cannot be contacted in case of emergency-   E 18. Subject is the investigator or any sub-investigator, research    assistant, pharmacist, study coordinator, or other staff thereof,    directly involved in the conduct of the protocol

Biological Status

-   E 19. Positive reaction to any of the following tests: hepatitis B    surface (HBs Ag) antigen, anti-hepatitis B core antibodies (anti-HBc    Ab) if compound having possible immune activities, anti-hepatitis C    virus (anti-HCV2) antibodies, anti-human immunodeficiency virus 1    and 2 antibodies (anti-HIV1 and anti HLIV2 Ab)-   E 20. Positive results on urine drug screen    (amphetamines/methamphetamines, barbiturates, benzodiazepines,    cannabinoids, cocaine, opiates)-   E 21. Positive alcohol test

Specific to the Study

-   E 22. Known hypersensitivity to insulin glargine and excipients-   E 23. Any history or presence of deep leg vein thrombosis or a    frequent appearance of deep leg vein thrombosis in first degree    relatives (parents, siblings or children)

Example 10: Treatments Investigational Product

-   -   Insulin glargine        -   Two different formulations of insulin glargine are used:            -   Lantus® U100 solution for injection containing 100 U/mL                insulin glargine (marketed product)            -   Insulin glargine U300 solution for injection containing                300 U/mL insulin glargine    -   Dose:        -   Lailtus® U100: 0.4 U/kg (=Reference R)        -   Insulin glargine U300: 0.4, 0.6 and 0.9 U/kg (=Test T₁-T₃)    -   Container: 3 mL glass cartridges    -   Route of application: Subcutaneously horizontally 5 cm right and        left of the umbilicus    -   Conditions: Fasted    -   Duration of treatment: 1 day at each period, single dose    -   Start: 09:00 on Day 1 (D1) in Treatment Periods 1 to 4 (TP1-4)    -   Additional treatments for 100% of included subjects are provided

TABLE 4 Treatments Reference treatment Test treatment Lantus ® U100Insulin glargine U300 INN Insulin glargine Insulin glargine (recombinanthuman (recombinant human insulin analogue) insulin analogue) Referencetreatment Test treatment Lantus ® U100 Insulin glargine U300 FormulationCartridges for 3 mL Cartridges for 3 mL solution U100 solution U300 1 mLcontains: 1 mL contains: 3.637 mg 21A-Gly- 10.913 mg 21A-Gly-30Ba-L-Arg-30Bb-L- 30Ba-L-Arg-30Bb-L- Arg human insulin Arg humaninsulin [equimolar to 100 [equimolar IU human insulin] to 300 IU humaninsulin] 30 μg zinc 90 μg zinc 2.7 mg m-cresol 2.7 mg m-cresol 20 mgglycerol 85% 20 mg glycerol 85% HCl and NaOH, pH 4.0 HCl and NaOH, pH4.0 specific gravity specific gravity 1.004 g/mL 1.006 g/mL Dose 0.4U/kg 0.4 U/kg 0.6 U/kg 0.9 U/kg Manufacturer sanofi-aventissanofi-aventis Deutschland GmbH Recherche & Development, Montpelier,France Batch commercial formulation, tbd purchased number through CROINN = international nonproprietary name

Dosing

This is a single dose study with in total 4 administrations of studymedication. Subjects are randomized to different sequences of thereference and test treatment such that each subject receives thereference treatment (R) and each of the test treatments (T₁₋₃) once.

Injections are given left or right of the umbilicus, with both sitesbeing used for separate injections. A washout period of 5 to 18 daysseparates consecutive dosing days, the preference is 7 days (7 daysbetween consecutive dosing). The length of the wash-out period variesindividually allowing both the participant and the investigator toadjust to their needs. By experience, 5 days comprise a minimum periodfor recovery enabling 1 clamp per week for a participant, while 18 daysrepresent a break of 3 weeks between dosing days, allowing subjects thefreedom to fulfill non-study related obligations, if unavoidable.

IP administration is administered under fasting conditions; subjectcontinues to fasten throughout the whole clamp period.

The blood glucose concentration is within a range of 5.5 mmol/L (100mg/dL)±20% without any glucose infusion for the last hour prior todosing during pre-clamp. When blood glucose has been stable for at least1 hour without any glucose infusion, IP is administered, IPadministration does not occur earlier than 09:00 clock time in themorning and not later than 14:00 clock time on Day 1 in TreatmentPeriods 1 to 4. If blood glucose is not stabilized before 14:00 hours,dosing does not occur. The visit is terminated and the subject isscheduled for a new dosing visit 1-7 days later.

Per subject and dosing a new cartridge is used.

IP administration is done by a person who is not otherwise involved inthe study or part of the study team at the CRO. This person gets therandom code to prepare IP administration in accordance to the openrandom list and doses subjects accordingly. The preparation and dosingis followed and checked by a second independent person. Respectivedocuments of dose preparation and treatment sequence is kept strictlyconfidential and is not being disclosed to any other person.

Calculation of Dose of IP (Insulin Glargine)

To calculate the amount of insulin glargine given for each subject, thebody weight (in kg) is determined to one decimal place and the amount ofinsulin calculated is rounded up or down to integer numbers as shown inthe following examples for a dose of 0.6 U/kg insulin glargine:

-   -   a subject with a body weight of 75.3 kg receives 45 U insulin        (75.3×0.6=45.18 which is rounded down to 45);    -   a subject with a body weight of 74.4 kg receives 45 U insulin        (74.4×0.6=44.64, which is rounded up to 45).

The body weight recorded during TP1 D1 is used for calculation of studymedication dose for all treatment periods. The study medication dose isnot to be changed if a subject's weight changes by less than or equal to2 kg between TP1 and one of the subsequent TPs. If a subject's bodyweight changes by more than 2 kg between TP1 and one of the subsequentTPs, the study medication dose is re-calculated based on the weight atD1 of the respective treatment period.

Syringes and Needles

Syringes with needles attached appropriate to accurately administersmall amounts of injection solution are used only (e.g. BectonDickinson, Ref 305502, Dimensions: 1 ML 27G ⅜ 0.40×10). The syringes aresupplied by the investigator.

Other Products

Other products used during the clamp procedure are described in Table 5.

TABLE 5 Preparation of infusion Manufac- Dose/Route of Drug Code INNFormulation turer administration Glucose Glucose 20% solution Certified,iv infusion for infusion selected by PROFIL Intramed Heparin VialCertified, iv infusion Heparin containing selected by Sodium 5 mLsolution PROFIL (5000 IU/mL) 0.9% Sodium Solution Certified, iv infusionSodium Chloride selected by Chloride PROFIL Apidra ® Insulin 100 U/mLfor sanofi- iv infusion glulisine injection aventis Glucose solution,sodium chloride solution, heparin and insulin glulisine is provided bythe Investigator. Glucose solution: 20% glucose solution is infused withthe Biostator ™ to keep subjects individual blood glucose at thedetermined target level. A second infusion pump (part of theBiostator ™) delivers 0.9% sodium chloride solution to keep the linepatent. In case the amount of 20% glucose solution needed exceeds theinfusion capacity of the Biostator ™, a second glucose infusion pump isengaged. Heparin: A low dose heparin solution (10.000 Units heparine/100mL saline) is infused via a double lumen catheter. The heparin solutionis taken up together with blood used for the Biostator's ™ blood glucosemeasurement in the other lumen of the catheter and is aimed to preventblood clotting in the system. Insulin glulisine: 15 U Apidra ® [100U/mL] is given to 49 mL of saline solution, to which 1 mL of thesubject's own blood is added to prevent adhesion, producing aconcentration of 0.3 U/mL, which is infused at an individual rate toachieve euglycemia.

Description of Blinding Methods

Subjects receive four different treatments (R, T₁, T₂ and T₃) in arandomized, blinded and crossover design.

In order to maintain the blinding, a third party un-blinded person isinvolved for IP dispensing and administration. This person is nototherwise involved in the study and/or part of the study team at theCRO, does not disclose any information to anyone and ensures to maintainblinding condition of the study. He/she gets the random code and dosessubjects accordingly. The preparation of IP and dosing is followed andchecked by a second independent person who has also access to the randomcode but is equally bound to confidentiality.

Method of Assigning Subjects to Treatment Group

IPs are administered according to the Clinical Study Protocol only tosubjects who have given written informed consent.

Subjects who comply with all inclusion/exclusion criteria are assignedjust before the Investigational Product administration on Day 1 inTreatment Period 1:

-   -   an incremental subject number according to the chronological        order of inclusion on the morning of D1 in Treatment Period 1.        The 9 digit subject number consists of 3 components (e.g. 276        001 001, 276 001 002, 276 001 003, etc.), of which the first 3        digits (276) are the country number, the middle 3 digits are the        site number and the last 3 digits are the subject incremental        number within the site. The subject number remains unchanged and        allows the subject to be identified during the whole study    -   a treatment number in a pre-planned order following the        randomized list with the next eligible subject always receiving        the next treatment number according to the randomization list

IP administration is in accordance with the randomized treatmentsequence.

Subjects withdrawn from the study retain their subject number and theirtreatment number, if already assigned. Replacement subjects have adifferent identification number (i.e., 500+the number of the subject whodiscontinued the study). Each subject receives the same treatmentsequence as the subject, who discontinued the trial

Screen Failed subjects are assigned a different number, e.g., 901, 902(to be recorded in the CRF only in case of AE occurring during screeningperiod after signing of informed consent).

Notes: The randomization of a subject occurs after Investigatorsconfirmation of subject's eligibility for this study. Baselineparameters are the parameters available the closest before the dosing.

Packaging and Labeling

Insulin glargine U300 solution is provided by sanofi-aventis inregrouping boxes of 3 mL cartridges.

The respective number of IP is packaged under the responsibility ofsanofi-aventis according to good manufacturing practice and localregulatory requirement and provided to CRO.

The content of the labeling is in accordance with the local regulatoryspecifications and requirements.

Lantus® U100 is commercially available and will be ordered by the CRO.

Storage Conditions

All IP is stored in an appropriate locked room under the responsibilityof the Investigator, and must be accessible to authorized personnelonly.

The IP has to be stored at +2° C. to +8° C., protected from light, andmust not be frozen.

Access to the randomization code during the study

In order to maintain the blinding, a third party un-blinded person isresponsible for IP dispensing and administration. This person is nototherwise involved in the study and/or part of the study team at theCRO, does not disclose any information to anyone and ensures to maintainblinding condition of the study. He/she gets the random code and dosessubjects accordingly. The preparation of IP and dosing is followed andchecked by a second independent person who has also access to the randomcode but is equally bound to confidentiality.

In case of an Adverse Event, the code is not being broken except in thecircumstances when knowledge of the Investigational Product is essentialfor treating the subject. For each subject, code-breaking material whichcontains the name of the treatment is supplied as envelopes. It is keptin a safe place on site throughout the Clinical Trial. The Sponsorretrieves all code-breaking material (opened or sealed) on completion ofthe Clinical Trial.

If the blind is broken, the Investigator documents the date of openingand reason for code breaking in the source data.

The Investigator, the clinical site pharmacist, or other personnelallowed to store and dispense IP is responsible for ensuring that the IPused in the study is securely maintained as specified by the Sponsor andin accordance with the applicable regulatory requirements.

All IP is dispensed in accordance with the Clinical Trial Protocol andit is the Investigator's responsibility to ensure that an accuraterecord of IP issued and returned is maintained.

Concomitant Treatment

The use of concomitant medication is not allowed during the study asspecified in Exclusion Criteria No. E14, with the exception of drugsmentioned there under, and is stopped within a given time frame (seeE14) before inclusion of the subject on Day 1 of Treatment Period 1.

To prevent interference of subjects' standard insulin treatment with theclamp measurement, subjects have to abstain from using basal insulinsand switch to

-   -   intermediate- or short-acting insulin products from 48 hours        prior to dosing at D1 of TP1 to TP4, if on long-acting insulin        products, i.e. Lantus® (insulin glargine), Levemir® (detemir) or        ultralente insulins,    -   short-acting insulins from 24 hours prior to dosing at D1 of TP1        to TP4 if on intermediate acting insulin products, i.e.        NPH-insulin

The last subcutaneous injection of short-acting insulin is no later than9 hours before study drug administration. Subjects on pump therapydiscontinues the insulin infusion in the morning of Day 1, at least 6hours prior to each IP administration (around 03:00 clock time assumingstart of IP administration at 09:00).

For symptomatic adverse events which are not jeopardizing the subjects'safety (e.g. headache) concomitant medication is reserved for adverseevents of severe intensity or of moderate intensity which persist for along duration. In particular, the use of acetaminophen/paracematol isprohibited if there is a known risk of hepatotoxicity, or as soon asabnormalities of liver enzymes occur.

However, if a specific treatment is required for any reason, an accuraterecord must be kept on the appropriate record form, including the nameof the medication (international nonproprietary name), daily dosage andduration for such use. The Sponsor must be informed within 48 h viae-mail or fax, with the exception of treatment of headache.

Treatment of potential allergic reactions will be in compliance with therecommendations as published elsewhere (Samspon H A, Munoz-Furlong A,Campbell R L et al. Second symposium on the definition and management ofanaphylaxis: summary report—Second National Institute of Allergy andInfectious Disease/Food Allergy and Anaphylaxis Network symposium.Journal of Allergy and Clinical Immunology 2006; 117(2):391-397).Dependent on the severity of the allergic reaction treatment withantihistamins, corticosteroids and epinephrine may be considered.

Treatment accountability and compliance

-   -   IP compliance:        -   IP is administered under direct medical supervision, and an            appropriate record is completed by the person responsible            for dispensing and administration of IP or his/her delegate;            any information on treatment sequence or dose is not            disclosed and documents are locked with no access by other            persons involved in the study        -   IP intake is confirmed by measurable drug assay results    -   IP accountability:        -   The person responsible for dispensing and administration of            IP or his/her delegate counts the number of cartridges            remaining in the returned packs, then fills in the Treatment            Log Form        -   The Investigator records the information about day and time            of dosing on the appropriate page(s) of the Case Report Form            (CRF)        -   The Monitor Team in charge of the study then checks the CRF            data by comparing them with the IP and appropriate            accountability forms after data base lock (to prevent            unblinding of the study)

Used cartridges are kept by the Investigator up to the fully documentedreconciliation performed with the Sponsor at the end of the study afterdata base lock.

Example 11: Assessment of Investigational Product

The present study is designed to assess the metabolic effect andexposure ratios of three different insulin glargine U300 doses versus0.4 U/kg Lantus® U100, to compare the duration of action of differentinsulin glargine U300 doses versus 0.4 U/kg Lantus® U100, to explore thedose response and dose exposure relationship of insulin glargine U300,and to asses the safety and tolerability of insulin glargine U300 in aneuglycemic clamp setting in subjects with diabetes mellitus type 1.

Pharmacodynamics Euglycaemic Clamp

The pharmacodynamic effect of insulin glargine, mainly the total glucosedisposal and duration of insulin action, is evaluated by the euglycemicclamp technique.

During the euglycemic clamp, arterialized venous blood glucoseconcentration, which reflects the supply for total glucose utilizationof all tissues, and the glucose infusion rate (GIR) needed to keep asubject's blood glucose concentration at its target level (clamp level)is continuously measured and recorded using the Biostator™ device(continuous glucose monitoring system, Life Sciences Instruments,Elkhart, Ind., USA).

The amount of glucose required (GIR-AUC) is a measure of the glucoseuptake into tissues (glucose disposal or glucose lowering activity)mediated by the exogenous insulin excess. The Biostator™ determinesblood glucose levels in 1 min intervals and adjusts the glucose infusionrate in response to changes in blood glucose using a predefinedalgorithm.

Clamp Procedure

To prevent interference of subjects' standard insulin treatment with theclamp measurement, subjects have to abstain from using basal insulinsand switch to

-   -   intermediate- or short-acting insulin products from 48 hours        prior to dosing at D1 of TP1 to TP4, if on long-acting insulin        products, i.e. Lantus® (insulin glargine), Levemir® (detemir) or        ultralente insulins,    -   short-acting insulins from 24 hours prior to dosing at D1 of TP1        to TP4 if on intermediate acting insulin products, i.e.        NPH-insulin

The last subcutaneous injection of short-acting insulin is no later than9 hours before IP administration. Subjects on pump therapy discontinuethe insulin infusion in the morning of Day 1, at least 6 hours prior toeach IP administration (around 03:00 clock time assuming start of IPadministration at 09:00).

During Treatment Periods 1 to 4 (TP1-TP4), subjects are admitted to theclinic in the morning of D1 after an overnight fast of at least 10hours.

In the morning of Day 1 the pre-clamp procedure starts and subjects arelinked to the Biostator™. Blood glucose concentration is adjusted to4.4-6.6 mmol/L (80-120 mg/dL) and maintained within these limits bymeans of iv bolus-administrations of a rapid acting insulin analog (e.g.insulin glulisine) and subsequent individual infusions of glucose asneeded.

60 min before study medication administration blood glucose is thenadjusted to 5.5 mmol/L (100 mg/dL)±20% (euglycemic clamp level) withoutany glucose infusion for the last hour prior to dosing. The insulinglulisine infusion is discontinued immediately prior to theadministration of the study medication.

When blood glucose has been stable for at least 1 hour within a range of5.5 mmol/L (100 mg/dL)±20% without any glucose infusion, IP isadministered (=T0 on D1 in TP1 to TP4, around 09:00). Subjects receivereference or test medication (R, T₁₋₃, see Table 4) as assigned byrandomization. Injections is given left or right of the umbilicus.

IP administration does not occur earlier than 09:00 clock time in themorning and not later than 14:00 clock time on Day 1 in TreatmentPeriods 1 to 4. If blood glucose is not stabilized during pre-clampbefore 14:00 clock time, dosing does not occur. The visit is terminatedand the subject is scheduled for a new dosing visit 1-7 days later.

IP administration is administered under fasting conditions; subjectcontinues to fasten throughout the whole clamp period.

The euglycemic clamp blood glucose level is continuously maintained bymeans of iv infusion of glucose solution until clamp end.

The goal of any basal insulin supplementation is to add to or even tosubstitute endogenous insulin secretion between meals. In subjectswithout endogenous insulin secretion, as invited to participate in thisstudy, exogenous insulin should provide for just the amount of insulinrequired to dispose hepatic glucose production. If perfectly matched,there is no need for extra glucose to compensate for excess insulin. Theresulting glucose infusion rate approximates zero. Once insulin actionceases, blood glucose concentration rises. The times to onset of riseand to times blood glucose concentrations exceeding predefinedthresholds are read by the Biostator™.

Selected doses of Lantus® U100 and insulin glargine U300 are above theaverage basal need which in turn produce some glucose demand reflectedin a sizeable GIR up to 36 hours.

The corresponding parameter indicative of the clamp performance, i.e.the precision for keeping blood glucose at clamp baseline level, is theblood glucose variability over the clamp period. A measure for bloodglucose variability is the coefficient of variation (CV %) perindividual clamp.

A low coefficient of variation in blood glucose is a prerequisite toproperly assess the insulin effect in clamp settings.

The clamp period is not to exceed 36 hours post study medicationinjection, the predefined clamp end.

Subjects continue fasting during the whole glucose clamp (pre-clamp andclamp) period while having access to water ad libitum.

In case blood glucose passes 11.1 mmol/L (200 mg/dL) prior to the clampend for 30 minutes after cessation of glucose infusion and theinvestigator confirms that any possible errors leading to false bloodglucose levels above 11.1 mmol/L (200 mg/dL) have been excluded, insulinglulisine used in the pre-IP administration time of the clamp is givento extend the observation period to 36 hours. In that case, the sponsorhas to be informed.

The subjects are delinked from the clamp setting when blood glucose iswell within the isoglycemic range.

Participants resume their pre-study medication on the day of dischargeat TP1 to TP4, i.e. Day 2.

The effect of the IPs is to last about 24-36 hours, which is why theparticipants is confined to the institute for 2 days.

A washout period of 5 to 18 days separates consecutive clamp perioddays, the preference is 7 days (7 days between consecutive dosing). Thelength of the wash-out period varies individually allowing both theparticipant and the investigator to adjust to their needs. Byexperience, 5 days comprise a minimum period for recovery enabling 1clamp per week for a participant, while 18 days represent a break of 3weeks between dosing days, allowing subjects the freedom to fulfillnon-study related obligations, if unavoidable.

Screening and D1 of TP1 is not separated by more than 28 days, while theEOS occurs no earlier than D5 or no later than D14 after last dosing,respectively.

Pharmacodynamic Sampling Times

Arterialized venous blood is continuously drawn at a rate of 2 mL/h fordetermination of arterial blood glucose concentration every minuteduring pre-clamp (prior to IP administration) and clamp period (up to 36hours after IP administration).

Arterialized venous blood samples (0.2 mL) for concurrent Biostator™calibration, which is a technical requirement, is collected at least in30 minute intervals after connection to the Biostator™ up to 36 hoursafter medication.

Number of Pharmacodynamic Samples

Blood glucose is continuously measured during the clamp procedure. Inaddition, at least 74 samples per subject and treatment period will becollected for calibration of the Biostator™ after IP administration. Intotal 74*4*24 samples or 7104 samples are collected (see table below).

TABLE 6 Number of blood samples and aliquots per subject during clampPeriods Glucose ^(a) Glucose ^(b) TP1 Continuously 74 TP2 Continuously74 TP3 Continuously 74 TP4 Continuously 74 Total number of Continuously296 samples per subject ^(a) continuous glucose monitoring at 2 mL/h forPD ^(b) calibration

Pharmacodynamic Handling Procedure

TABLE 7 Sample Handling Procedures Blood Sample Analyte Volume HandlingProcedures Glucose for PD 2 mL/h none Glucose for 200 μL Blood to befilled calibration into capillary and then into sample cup for immediateanalysis

Pharmacodynamic Parameters

The area under the body weight standardized GIR within 36 hours(GIR-AUC₀₋₃₆) and the time to 50% of the total GIR-AUC within 36 hours(T_(50%)-GIR-AUC₀₋₃₆) is calculated.

Duration of blood glucose control is taken as the time in euglycemiafrom dosing to deviation above clamp glucose level (100 mg/dL). Times ofcontrolled blood glucose within predefined margins is taken from dosingto specified thresholds, e.g. blood glucose levels at 110, 130 and 150mg/dL.

In addition, the maximum smoothed body weight corrected GIR (GIR_(max))and the time to GIR_(max), GIR-T_(max), is assessed.

Further supplemental parameters is derived as appropriate.

Safety Baseline Demographic Characteristics

The baseline demographic characteristics consists of:

-   -   Age (years)    -   Body weight (kg)    -   Height (cm)    -   Body Mass Index (BMI) (kg/m²)

Safety Assessment at Baseline and During the Study

-   -   Physical examination at screening: cardiovascular system, chest        and lungs, thyroid, abdomen, nervous system, skin and mucosae,        and musculo-skeletal system and relevant medical and surgical        history, diabetes history (diagnosis of diabetes, onset of        insulin treatment, late complications); only findings relevant        to the study are documented    -   Past and current smoking status    -   Physical examination at pre-dose and during the study:        cardiovascular system, abdomen and lungs; only findings relevant        to the study are documented    -   Body temperature (aural)    -   Vital signs: Heart rate, respiratory rate and systolic and        diastolic blood pressure measured after 10 minutes in supine        resting position, heart rate and systolic and diastolic blood        pressure—also after 3 minutes in standing position (except for        unscheduled measurements when connected to Biostator™)

Laboratory tests (in fasted conditions for blood samples):

-   -   Hematology: Red blood cell count (RBC), hematocrit (Hct),        hemoglobin (Hb), white blood cell count (WBC) with differential        (neutrophils, eosinophils, basophils, monocytes and        lymphocytes), platelets, INR and aPTT    -   Biochemistry:        -   Electrolytes: Sodium, potassium, bicarbonate, chloride,            calcium        -   Liver function: AST, ALT, alkaline phosphatase,            gamma-glutamyl transferase (γGT), total and conjugated            bilirubin        -   Renal function: creatinine, BUN        -   Metabolism: Glucose, albumin, total proteins, total            cholesterol, triglycerides, HbA1c (at screening, D1 TP1 and            EOS), LDH, amylase, lipase, C-peptide (screening only)        -   Potential muscle toxicity: Creatinine phosphokinase (CPK)        -   Serology: Hepatitis B antigen (HBs Ag), anti-hepatitis B            core antibodies (anti-HBc Ab), anti-hepatitis C antibodies            (anti-HCV2), anti-HIV1 and anti-HIV2 antibodies    -   Archival blood sample: a 5 mL blood sample is collected into a        dry, red topped tube, centrifuged at approximately 1500 g for 10        minutes at 4° C.; the serum is then transferred into three        storage tubes, which are immediately capped and frozen in an        upright position at −20° C. This sample is used if any        unexpected safety issue occurs to ensure that a pre drug        baseline value is available for previously non-assessed        parameters (e.g., serology). If this sample is not used, the        Investigator destroys it after the Sponsor's approval    -   Urinalysis: Proteins, glucose, blood, ketone bodies, pH        -   Qualitative: A dipstick is performed on a freshly voided            specimen for qualitative detection using a reagent strip;        -   Quantitative: A quantitative measurement for glucose,            protein, erythrocytes and leucocytes count is required in            the event that the urine sample test is positive for any of            the above parameters by urine dipstick (e.g., to confirm any            positive dipstick parameter by a quantitative measurement).    -   Urine drug screen: Amphetamines/metamphetamines, barbiturates,        benzodiazepines, cannabinoids, cocaine, opiates    -   Alcohol breath test    -   Pregnancy/hormone test (if female):        -   β-HCG in blood at screening        -   urine β-HCG at TP1 to TP4, Day 1        -   FSH/estradiol, if postmenopausal less than 2 years, at            screening only    -   Adverse Events: Spontaneously reported by the subject or        observed by the Investigator    -   ECG telemetry (single lead)    -   12-lead ECG (automatic)    -   Anti-insulin antibodies

Blood samples for laboratory tests are taken under fasted conditions.

ECG Methodology ECG Telemetry

-   -   ECG telemetry is continuously monitored by medical personnel.        All arrhythmic events will be documented by printing and        included in the subject's CRF. This documentation allows for        diagnosis of the event, time of occurrence, and duration, and is        signed by the Investigator or delegate. The ECG telemetry        records is kept for a potential re-analyze taking account the        Investigational Product exposure.

Twelve-Lead ECGs

-   -   Twelve-lead ECGs are recorded after at least 10 minutes in        supine position using an electrocardiographic device (MAC        5500™). The electrodes are positioned at the same place for each        ECG recording throughout the study (attachment sites of the        leads are marked with an indelible pen).    -   ECGs is always recorded before the PK sampling (if any). PK        samples are drawn as soon as possible (within 15 minutes) after        ECG.    -   Each ECG consists of a 10 second recording of the 12 leads        simultaneously, leading to:        -   a single 12-lead ECG (25 mm/s, 10 mm/mV) print-out with HR,            PR, QRS, QT, QTc automatic correction evaluation, including            date, time, initials and number of the subject, signature of            the investigator, and at least 3 complexes for each lead.            The Investigator medical opinion and automatic values is            recorded in the CRF. This print-out is retained at the site            level        -   a digital storage that enables eventual further reading by            an ECG central lab: each digital file is identified by            theoretical time (day and time DxxTxxHxx), real date and            real time (recorder time), Sponsor study code, subject            number (i.e., 3 digits) and site and country numbers if            relevant.    -   The digital recording, data storage and transmission (whenever        requested) comply with all the applicable regulatory        requirements (i.e., FDA 21 CFR, part 11).

When vital signs, ECG, and blood samples are scheduled at the same timeas an Investigational Product administration and/or a meal, they aredone prior to Investigational Product administration and/or meal.Whenever measurements of vital signs, ECG, and blood samples for PK, PD,or safety coincide, the following order is respected: ECG, vital signs,PD, PK, and safety samples; in order to respect exact timing of PKsamples (refer to flow-chart for time window allowance for PK samples),the other measures are done ahead of the scheduled time.

The assessment schedule is adapted to the design of the study

Local Tolerability at Injection Site

Findings at the site of injection (such as erythema, edema, papules,induration, vesicles, blisters) are graded mainly according to a GlobalIrritation Score. A local injection site reaction with a score of ≥3according to the rating scale is documented additionally as an adverseevent.

The subjects are asked to report sensations at the injection site.

Pharmacokinetics

For the assessment of insulin glargine pharmacokinetics, the area underthe insulin concentration curve (INS-AUC) up to 36 hours, INS-AUC₀₋₃₆and the time to 50% of INS-AUC₀₋₃₆ is derived. In addition, the maximuminsulin concentration INS-C_(max), and time to C_(max) (INS-T_(max)) isobtained.

Sampling Times

Blood is collected for the determination of insulin glargineconcentrations at time points OH, 1H, 2H, 4H, 6H, 8H, 12H, 16H, 20H,24H, 28H, 32H and 36H after injection of study medication.

Number of Pharmacokinetic Samples

TABLE 8 Number of blood samples per subject Insulin Periods (glargine)Treatment Period 1 13 Treatment Period 2 13 Treatment Period 3 13Treatment Period 4 13 Total number of samples per subject 52 Totalnumber of samples ^(a) 52*24 = 1248 ^(a) assuming 24 subjects completedthe study

Pharmacokinetic Handling Procedure

The exact time of IP administration and sample collection must berecorded in the CRF.

Pharmacokinetic Parameters

The following pharmacokinetic parameters are calculated, usingnon-compartmental methods for insulin glargine concentrations aftersingle dose. The parameters include, but are not be limited to thefollowing.

TABLE 9 List of pharmacokinetic parameters and definitions ParametersDrug/Analyte Definition/ Calculation C_(max) Insulin Maximumconcentration observed T_(max) Insulin First time to reach C_(max)AUC₀₋₃₆ Insulin Area under the concentration versus time curvecalculated using the trapezoidal method from time zero to 36 hours postdosing T_(50%)-AUC Insulin Time to 50% of AUCq-36

Sampled Blood Volume

TABLE 10 Sampled Blood Volume Volume Sample Type per Sample Number TotalSerology 2 mL 1 2 mL Hematology 2.7 mL 5 13.5 mL Coagulation 2 mL 3 6 mLBiochemistry 5 mL 3 15 mL Archival Sample 5 mL 1 5 mL Insulin 3 mL 13*4156 mL Glucose calibration 0.2 mL 74*4 59.2 mL Glucose continuously 2mL/h 40*4 320 mL [3-HCG (if female) ^(a) 0 mL 1 0 mL FSH/estradiol (iffemale) ^(a,b) 0 mL 1 0 mL Anti-insulin antibodies 3 mL 2 6 mL Total582.7 mL ^(a) included in serology ^(b) if postmenopausal less than 2years

Measures to Protect Blinding of the Trial

In order to maintain the blinding, a third party un-blinded person isinvolved for IP dispensing and administration. This person is nototherwise involved in the study and/or part of the study team at the CROor sponsor. He/she gets the random code provided by sanofi-aventis anddoes not disclose the random code or any other information to any otherperson. For safety reason, the treatment randomization code is unblindedfor reporting to the Health Authority of any Suspected UnexpectedAdverse Drug Reaction (SUSAR) and reasonably associated with the use ofthe IP according to either the judgment of the Investigator and/or theSponsor.

Subject Safety

The Investigator is the primary person responsible for taking allclinically relevant decisions in case of safety issues.

If judged necessary, the opinion of a specialist should be envisaged ina timely manner (e.g. acute kidney failure, convulsions, skin rashes,angioedema, cardiac arrest, electrocardiographic modifications, etc).

Example 12: Study Procedures Visit Schedule Screening Procedures

Screening procedures are carried out within 28 days up to 3 days priorto inclusion to determine subject's eligibility for participation. Thesubject receives information on the study objectives and procedures fromthe Investigator. The subject signs the informed consent prior to anyaction related to the study. Recording of adverse events startsthereafter.

Prior to screening, subjects have fasted (apart from water) for 10 hours(excluding a small amount of carbohydrates as countermeasure forhypoglycemia, if necessary).

The screening visit includes the following investigations:

-   1 Demographics (age, sex, race, past and current smoking status,    height, body weight, BMI)-   2 Physical examination (cardiovascular system, chest and lungs,    thyroid, abdomen, nervous system, skin and mucosae, and    musculo-skeletal system) and relevant medical and surgical history,    diabetes history (diagnosis of diabetes, onset of insulin treatment,    late complications); only findings relevant to the study are    documented-   3 Relevant previous and all concomitant treatments, average insulin    regimen in the last 2 months prior to study entry-   4 ECG (standard 12 lead), vital signs measurements (pulse rate,    systolic and diastolic blood pressure measured after 10 minutes in    supine resting position, and after 3 minutes in standing position),    and core body temperature (aural)-   5 Laboratory tests with hematology, HbA1c, C-peptide, clinical    chemistry, serology, urinalysis, urine drug screen, alcohol breath    test, β-HCG and FSH/estradiol blood test (female only, if    applicable)

One retest within a week is permitted with the result of the last testbeing conclusive.

Subjects who meet all the inclusion criteria, and none of the exclusioncriteria, are eligible for the inclusion visit.

In case of screening failures the basic results of the screeningexamination are recorded in the source documents.

Inclusion procedures (Day 1 of Treatment Period 1)

Subjects, who qualify for enrollment into the study, are admitted to theclinic in the fasted state in the morning of D1 of TP1 at approximately07:00.

The inclusion examination is carried out on the first dosing day (D1,TP1) and includes the following investigations:

-   Physical examination with updated medical history (AEs),    previous/concomitant medication and aural body temperature-   Body weight, BMI (height measured at screening)-   ECG (standard 12 lead), vital signs measurements (heart rate,    respiratory rate, systolic and diastolic blood pressure measured    after 10 minutes in the supine resting position, and after 3 minutes    in the standing position)-   Laboratory tests with hematology, clinical chemistry, urinalysis,    urine drug screen, alcohol breath test, β-HCG urine test (female    only, if applicable).

Each subject receives an incremental identification number according tothe chronological order of his/her inclusion in the study.

Randomization occurs on D1/TP1 after confirmation of subject'seligibility by the Investigator. If more than one subject is randomizedat the same time, subjects are randomized consecutively according to thechronological order of inclusion on the morning of Day 1/TP1, i.e. thesubject with the lowest subject number receives the next availablerandomization number.

Results of laboratory tests of D1/TP1 are baseline values and consideredconfirmatory, with the exception of the β-HCG urine test (based onsample collected during screening visit), which must be negative.

If a subject is finally enrolled, a blood sample is taken for archivingand for determination of anti-insulin antibodies (on D1/TP1 only).

Description by Type of Visit Treatment Periods 1-4 (D1 to D2)

To prevent interference of subjects' standard insulin treatment with theclamp measurement, subjects abstain from using basal insulins and switchto

-   -   intermediate- or short-acting insulin products from 48 hours        prior to dosing at D1 of TP1 to TP4, if on long-acting insulin        products, i.e. Lantus® (insulin glargine), Levemir® (detemir) or        ultralente insulins,    -   short-acting insulins from 24 hours prior to dosing at D1 of TP1        to TP4 if on intermediate acting insulin products, i.e.        NPH-insulin

The last subcutaneous injection of short-acting insulin is no later than9 hours before IP administration. Subjects on pump therapy discontinuesthe insulin infusion in the morning of Day 1, at least 6 hours prior toeach IP administration (around 03:00 clock time assuming start of IPadministration at 09:00).

Upon arrival at the clinic, subjects are asked to ensure that they havehad no clinically relevant changes in their physical condition since theprevious visit, that they have been compliant with the general anddietary restrictions as defined in the protocol and that they changedtheir insulin treatment, if required. Violation of the study criteriaexcludes the subject from further participation in the study. Dependingon the kind of violation, a subject may be excluded only from theparticular study day, allowing a re-scheduling of the study day once, orfor the entire study.

Any changes in the health condition and the concomitant medication ofthe subjects since the last visit are reported in the subject's medicalrecords (source) and the CRF.

In the morning shortly before administration of study medication (D1 ofeach TP) body weight, vital signs, 12-lead ECG, ECG monitoring and corebody temperature are recorded, an urinalysis and a urine drug andalcohol screen are performed.

The amount of insulin glargine required for injection will be calculatedaccording to subject's body weight.

Hematology is analyzed for incurring anemia on Day 1 of Treatment Period3. If positive, the wash-out interval between Treatment Periods 3 and 4is extended to the maximum allowed 18 days or start of TP4 will bepostponed until hematological parameters have been normalized. Anadditional hematological assessment is made on Day 1 of Treatment Period4.

Subjects remains fasting (apart from water) until the end of theeuglycemic clamp.

Subjects are then be prepared for the start of the pre-clamp procedurewith three venous lines connected to an automatic glucose reading device(Biostator™) and remain in semi-recumbent position for the entireduration of the sampling period. At approximately 07:30 a dorsal handvein or lateral wrist vein of the left arm is cannulated and connectedto the Biostator™ in order to continuously draw arterialized venousblood for the determination of blood glucose concentration. The lefthand is placed into a heated box (“Hot-Box”), which provides for an airtemperature of about 55° C., allowing arterialization of venous blood. Asecond venous line is placed into the antecubital vein of the left armand is used to collect samples for insulin and reference blood glucosedetermination. A third vein is cannulated on the contralateral forearmallowing the infusion of 0.9% saline and 20% glucose solution with apump in the Biostator™ or insulin glulisine with an external pump.

From insertion of the vascular catheters until 60 min before studymedication administration at approximately 09:00 on D1, the bloodglucose level is maintained within 4.4 to 6.6 mmol/L (80-120 mg/dL,pre-clamp). Depending on the blood glucose level, additional intravenousbolus injection of insulin glulisine is given to keep the blood glucosewithin the target range. In the 1 hour before study medicationadministration no intravenous bolus injections are given until clampend.

Additional blood samples for the determination of blood glucose aretaken in at least 30 min intervals to check against a laboratoryreference based on the glucose oxidase method. If necessary theBiostator™ is re-calibrated according to results of the laboratoryreference method.

Insulin infusion rates are adjusted individually. While keeping bloodglucose at the target level both, insulin and glucose infusion rate areminimized during the clamp run-in phase. Insulin glulisine solution isinfused by means of a high precision infusion pump (Terumo SpritzenpumpeTE 311™), 20% glucose solution is be applied by a high precisioninfusion pump (Terumo Infusionspumpe TE 171™).

The clamp level is adjusted 60 min before study medicationadministration to maintain the blood glucose at about 5.5 mmol/L (100mg/dL) until the end of the clamp period. The pre-clamp is prolonged andIP administration postponed until 14:00 clock time in case the targetglucose level has not been met during the run-in phase (pre-clamp). Ifthe target glucose level cannot be established within until 14:00 clocktime, the visit is terminated and the subject may be scheduled for a newdosing visit 1-7 days later.

The insulin glulisine infusion is discontinued immediately before studymedication administration. The first insulin sample for PK is takenimmediately thereafter. At about 09:00 the study medication isadministered (Table 4), either

-   -   the Reference treatment (R, 0.4 U/kg Lantus® U100)    -   or the Test treatment (T₁₋₃) at one peri-umbilical site        according to the randomization plan, using a standardized        skin-fold technique.

During the clamp 12-lead ECGs are taken 2 and 12 hours after injectionof IP and at clamp end.

The study medication is administered preferably by the same person atduring the whole study.

The end of the injection defines time zero (T0), which defines thestarting time of the subsequent clamp period and PK sampling.

Every clamp observation period lasts 36 hours and thus ends atapproximately at 21:00 on D2, the predefined end-of-clamp. Thereafterthe subjects are delinked from the euglycemic clamp setting when bloodglucose is well within the isoglycemic range, receive a meal and theirusual insulin treatment.

In case blood glucose passes 11.1 mmol/L (200 mg/dL) during the clampperiod for 30 minutes after cessation of glucose infusion and theinvestigator confirms that any possible errors leading to false bloodglucose levels above 11.1 mmol/L (200 mg/dL) have been excluded, therapid acting insulin analog (e.g. insulin glulisine) used in the pre-IPadministration time of the clamp is given to extend the clamp period to36 hours for pharmacokinetic blood sampling. In that case, the sponsorhas to be informed. Thereafter the subjects are delinked from theeuglycemic clamp setting when blood glucose is well within theisoglycemic range, receive a meal and their usual insulin treatment.

The injection site reaction is assessed 15 minutes as well as one hourafter injection of the study medication and documented as an AE if ascore of >3 is observed according to the rating scale.

Prior to discharge, a meal ad libitum is served and the usualinsulin-treatment will be resumed. Vital signs (heart rate; systolic anddiastolic blood pressure measured after 10 minutes in the supine restingposition, and after 3 minutes in the standing position) are repeated andblood glucose is measured (the blood glucose reading must be above 80mg/dL). Subjects are discharged on D2 of TP1 to TP4 after theirwell-being is ensured by the investigator.

End-of-Study Visit

Subjects return for an end-of-study (EOS) visit between D5 and D14 afterlast dosing in TP4.

Subjects have fasted (apart from water) for 10 hours. The EOS includesthe following investigations:

-   Physical examination (weight, body temperature) with updated medical    history-   ECG, vital signs measurement-   Laboratory tests with hematology, HbA1c, biochemistry, urinalysis,    and if female a β-HCG blood test-   Any AE occurred or concomitant medication taken since TP4-   Blood sample for anti-insulin antibody determination.

The Investigator ensures that based on all available clinical results,the subject can be safely released from the study.

Study Restriction(s)

Subjects ceases their usual insulin treatment on Days-2 to -1, dependingon the type of insulin used (long acting, NPH, intermediate).Thereafter, the blood glucose levels are controlled solely by multiplesubcutaneous injections of the usual short-acting insulin.

The usual insulin treatment is resumed after discharge on Day 2 in TP1to TP4.

The subjects do not take any concomitant medication, which willinterfere with the metabolic control or the insulin sensitivity ofsubjects throughout the study and in the two weeks before the study.

Consumption of alcoholic beverages, grapefruit juice, and stimulatingbeverages containing xanthine derivatives (tea, coffee, Coca Cola-likedrinks, chocolate) is not permitted 24 hours before administration ofeach study medication until the end of the clamp.

Orange juice or similar carbohydrates are given as corrective measuresfor hypoglycemia during clamp if not adequately counteracted byintravenous glucose infusion when connected to the Biostator™.

No strenuous physical activity is allowed within 2 days before eachstudy medication administration.

Subjects who smoke 5 or less cigarettes per day are included in thestudy and subjects may smoke during the study, except on D1 and D2 ofTP1 to TP4.

On the screening day, subjects come to the unit after an overnight fastof at least 10 hours (excluding a small amount of carbohydrates ascountermeasure for hypoglycemia, if necessary).

In the morning of Day 1 in TP1 to TP4, subjects are admitted to theclinic after an overnight fast of at least 10 hours and remain fastinguntil end of clamp period in Day 2. A meal ad libitum is served afterthe end of the clamp.

Fluid supply is at least 2500 mL for each 36-hour period.

Definition of Source Data

All evaluations listed below that are reported in the CRF are supportedby appropriately signed identified source documentation related to:

-   -   subject identification    -   medical history (in case of allergic reaction)    -   clinical examination, vital signs, body weight and height, body        temperature;    -   laboratory assessments, ECG    -   pharmacokinetic time points    -   dates and times of visits and assessments    -   adverse events    -   IP administration    -   previous/concomitant medication    -   start/end of clamp procedure, clamp data

Example 13: Statistical Considerations Determination of Sample Size

The primary objective of the study is to assess the relative metaboliceffect for insulin glargine given as one dose of U100 (R) and threedifferent doses of U300 (T₁ to T₃).

Based on the data of study PKD10086, a value of approximately 0.375 canbe expected for the SD_(within) of GIR-AUC_(end of clamp) on the naturallog-transformed scale.

For the purpose of the sample size calculation within-subject SDsbetween 0.325 and 0.425 were used.

Table 11 shows the maximum imprecision (in terms of the 90% confidenceinterval) for a pairwise treatment ratio of adjusted geometric meansthat will be obtained with 90% assurance, for total number of subject Nbetween 16 and 24, assuming a true within-subject SD of values between0.325 and 0.425 for log GIR-AUC₀₋₃₆.

TABLE 11 Maximum imprecision for any pairwise ratio Confidence level:90% Assurance: 90% Within- Total Maximum Maximum width 90% CI subject SDnumber imprecision for an observed ratio equal to on log scale ofsubjects (%) 0.6 0.8 1 0.325 16 19.7 (0.48; 0.75) (0.64; 1.00) (0.80;1.25) 20 17.5 (0.49; 0.73) (0.66; 0.97) (0.82; 1.21) 24 15.9 (0.50;0.71) (0.67; 0.95) (0.84; 1.19) 0.350 16 21.0 (0.47; 0.76) (0.63; 1.01)(0.79; 1.27) 20 18.7 (0.49; 0.74) (0.65; 0.98) (0.81; 1.23) 24 17.0(0.50; 0.72) (0.66; 0.96) (0.83; 1.21) 0.375 16 22.4 (0.47; 0.77) (0.62;1.03) (0.78; 1.29) 20 19.9 (0.48; 0.75) (0.64; 1.00) (0.80; 1.25) 2418.1 (0.49; 0.73) (0.65; 0.98) (0.82; 1.22) 0.400 16 23.7 (0.46; 0.79)(0.61; 1.05) (0.76; 1.31) 20 21.1 (0.47; 0.76) (0.63; 1.01) (0.79; 1.27)24 19.2 (0.48; 0.74) (0.65; 0.99) (0.81; 1.24) 0.425 16 24.9 (0.45;0.80) (0.60; 1.07) (0.75; 1.33) 20 22.3 (0.47; 0.77) (0.62; 1.03) (0.78;1.29) 24 20.3 (0.48; 0.75) (0.64; 1.00) (0.80; 1.25)

With 20 subjects, if the true within-subject SD of GIR-AUC₀₋₃₆ is asmuch as 0.375, the treatment ratio will be estimated with a maximumimprecision of 19.9% (i.e. the 90% CI will be 0.80 and 1/0.80=1.25 timesthe observed ratio), with 90% assurance.

24 subjects will be included in order to have 20 completed subjects

Subject Description Disposition of Subjects

A detailed summary of subject accountability including count of subjectsincluded, randomized, exposed (i.e. received any amount of studymedication), completed (i.e. subjects who completed all study treatmentperiods), discontinued along with the main reasons for discontinuationis generated.

Subject disposition at the final visit is presented in a listingincluding sequence group, disposition status at the end of the studywith the date of last administration of study drug, date of final visit,reason for discontinuation. All withdrawals from the study, taking placeon or after the start of the first study drug administration, are fullydocumented in the body of the clinical study report (CSR).

Protocol Deviations

Prior to data lock of the study, Clinical Trial Protocol deviations areexamined relative to criteria defined for definition of populations andother study criteria including:

-   -   Inclusion and exclusion criteria;    -   Treatment compliance;    -   Compliance with the Clinical Trial Protocol with regard to        prohibited therapies;    -   Compliance with the Clinical Trial Protocol with regard to        intervals between visits and total treatment duration; and    -   Whether planned activity and safety evaluation were performed,        etc.

Deviations covered include but not be limited to:

-   -   Subjects without any evaluation (of any variables) after        randomization;    -   Subjects not exposed;    -   Subject without any evaluation of the primary variable (if        relevant);    -   Subjects who entered the study even though they did not satisfy        the inclusion criteria;    -   Subjects who developed withdrawal criteria during the study but        were not withdrawn;    -   Subjects who received the wrong treatment or incorrect dose;    -   Subjects who received a prohibited concomitant medication.

Major deviations are listed and summarized.

Analysis Population

All exclusions from any analysis populations (pharmacodynamic,pharmacokinetic and/or safety) are fully documented in the CSR.

Subjects excluded from any analysis population are listed with treatmentsequence, and with reason for exclusion. Any relevant information isfully documented in the CSR. Frequencies of subjects, overall and pertreatment, for the analysis populations are tabulated.

For the event of subjects having received treatments that differed fromthose assigned according to the randomization schedule, analyses areconducted according to the treatment received rather than according tothe randomized treatment.

Pharmacodynamic Population

All subjects without any major deviations related to study drugadministration, and for whom PD parameters are available, are includedin the pharmacodynamic population. For subjects with insufficient PDprofiles in one but not both treatment periods, parameters of thesufficient profiles are included in the analysis.

For subjects, who receive (for safety reasons) insulin glulisine withinthe observation period of 36 hours after dosing of IP, pharmacodynamicdata are only taken into account up to the time of administration ofinsulin glulisine.

Exclusions from Pharmacodynamic Analysis

All exclusions form the pharmacodynamic analysis are listed togetherwith the reason. Exclusions are decided and documented based on thereview of the data prior to database lock and unblinding.

Safety Population

All subjects who were exposed to any comparative study treatment,regardless of the amount of treatment administered, are included in thesafety population.

Pharmacokinetic Populations

All subjects without any major deviations related to study drugadministration, and for whom insulin PK parameters are available, areincluded in the pharmacokinetic population. For subjects withinsufficient insulin PK profiles at one but not all treatment periods,parameters of the sufficient profiles are included in the analysis.

The bioanalytical assay for insulin glargine is interfered by otherinsulins like insulin glulisine. Therefore, the pharmacokinetic data forinsulin glargine of those subjects are excluded from evaluation, whohave received (for safety reasons) insulin glulisine within the clampobservation period of 36 hours after IP administration.

Demographic and Baseline Characteristics Subject DemographicCharacteristics, Medical History and Diagnoses

The following data are collected: sex, age, height, weight, and race.Baseline body mass index (BMI) per subject is calculated from pre-dosebody weight and height data:

BMI=body weight [kg]/(height [m])²

All variables concerning demographic and background characteristics arelisted individually and summarized for the safety population.

Deviations from inclusion criteria related to medical history anddiagnoses are listed and described individually.

Baseline Safety Parameters

For safety variables, the latest scheduled value before study drugadministration within the period or within the study, whatever isapplicable for the variable, is taken as the baseline value. If thebaseline pre-dosing value is rechecked before dosing, the recheckedvalue is considered as the baseline and used in statistics.

Extent of study treatment exposure and compliance

Details of study drug dosing and complementary information are listedindividually and summarized if appropriate.

Individual total doses of insulin glargine are summarized by treatment.

Prior/Concomitant medication/therapy

Prior and concomitant medications/therapies (if any) are coded accordingto the World Health Organization-Drug Reference List (WHO-DRL, latestversion in use at time of database lock) and are listed individually.

Concomitant insulin medication (subcutaneous) is listed separately.

Insulin infusion or bolus given at any time during the clamp procedureis listed or plotted over time on an individual basis. Insulin infusionor bolus given after dosing during the clamp procedure is listed on anindividual basis.

Analysis of Pharmacodynamic Variables

All pharmacodynamic analyses encompass data of the pharmacodynamicpopulation. No adjustment of the alpha-level is made for multipleanalyses.

For pharmacodynamics of insulin glargine, the blood glucoseconcentration and glucose infusion rate (GIR) is continuously recordedduring the clamp procedure.

Statistical analyses compare test treatments (T₁ to T₃) with thereference treatment (R)

Description of Pharmacodynamic Variables

In order to achieve comparability between the subjects body weightadjusted insulin dosing, all values for GIR are divided by the subject'sbody weight in kg for analysis. Thus in the below, if not statedotherwise, GIR always refers to the body weight standardized glucoseinfusion rate.

Primary PD Variable

The following PD variable is considered primary.

-   -   Area under the body weight standardized glucose infusion rate        time curve [GIR-AUC₀₋₃₆ (mg/kg)]

GIR-AUC₀₋₃₆ is calculated according to the rectangular rule for thestepwise constant function with timescale in minutes.

Secondary PD Variables

The following PD variables are derived and considered secondary:

-   -   Time (h) to 50% of GIR-AUC₀₋₃₆ [T_(50%)-GIR-AUC₀₋₃₆ (h)]    -   Maximum smoothed body weight standardized glucose infusion rate        [GIR_(max) (mg*min/kg)]    -   First time after dosing to reach GIR_(max) [GIR-T_(max)(h)]    -   Duration of euglycemia (time to elevation of smoothed blood        glucose profile above clamp level) is calculated as the time        from dosing to the last value of the smoothed blood glucose        concentration curve at or below 105 mg/dL    -   Durations of controlled blood glucose within predefined margins        are defined as the time from dosing to the last value of the        smoothed blood glucose concentration curve at or below        -   110 mg/dL        -   130 mg/dL        -   150 mg/dL

Smoothing

The maximum of the raw body weight standardized GIR is subject to thenoise in the GIR adjustment. Thus, the derivation of GIR_(max) and thetime to GIR_(max), is based upon a LOESS (locally weighted regression insmoothing scatterplots) smoothing technique for the raw body weightstandardized GIR data. Due to the expected morphology of theGIR-profiles as known under Lantus®, a smoothing factor of 6% is used(SAS®, PROC LOESS, factor 0.06).

Blood glucose levels are well be subject to noise. Therefore, theduration of euglycemia and the duration of blood glucose control arebased upon a LOESS (locally weighted regression in smoothingscatterplots) smoothing technique for the raw blood glucose levels. Dueto the expected morphology, a smoothing factor of 6% is used (SAS®, PROCLOESS, factor 0.06).

In case of inadequate smoothing a different smoothing factor is used foran additional analysis.

Additional PD Variables

Further parameters are derived, as:

-   -   Time to end of glucose infusion, as the latest time after dosing        with GIR above zero

Additional PD variables are derived if deemed necessary forinterpretation of results.

Primary PD Analysis

Prior to the analysis described below, GIR-AUC₀₋₃₆ is log-transformed(natural log).

Log-transformed GIR-AUC₀₋₃₆ is analyzed with a linear mixed effectsmodel with fixed terms for sequence, period and treatment

log(parameter)=sequence+period+treatment+error

and with an unstructured R matrix of treatment (i, i) variances andcovariances for subject within sequence blocks, using SAS PROC MIXED.

90% confidence interval (CI) for the ratio of treatments geometric means(T₁/R, T₂/R, T₃/R) is obtained by computing estimate and 90% CI for thedifference between treatment means within the linear mixed effects modelframework, and then converting to ratio of geometric means by theantilog transformation. Equivalence is concluded if the 90% CI for theratio is entirely within the 0.80 to 1.25 equivalence referenceinterval.

Listings of individual ratios (test treatments versus referencetreatment) are provided with the corresponding descriptive statistics.

Secondary Analysis/Analysis of Secondary Variables DescriptivePresentations for GIR Profiles

Individual body weight standardized GIR (mg*min/kg) is plotted for raw,smoothed and cumulative raw values.

Mean and median body weight standardized GIR-profiles as well as medianpercentage cumulative profiles over time are plotted by treatment.

Cumulative plots cover the time between dosing to end of clamp.

Descriptive Presentations for Derived PD Parameters

PD parameters are listed individually, and descriptive statistics aregenerated by treatment.

Treatment Ratios for Secondary PD Parameters

Treatment ratios (T₁/R, T₂/R, T₃/R) with confidence limits are derivedfor maximum standardized glucose infusion rate [GIR_(max)(mg*min/kg)]using the corresponding linear mixed effects model as described abovefor the primary analysis. Exploratory comparisons between treatments arebased on conventional bioequivalence criteria (90% confidence limits0.80 to 1.25).

The distribution of GIR-T_(max) values is represented by histogram plotsfor each treatment. In addition, a histogram of differences inGIR-T_(max) between test treatments and reference is provided.

Treatment Differences for Secondary PD Parameters

T_(50%)-GIR-AUC₀₋₃₆ (h) is analyzed non-parametrically based onHodges-Lehmann method for paired treatment comparisons. CIs forpair-wise treatment differences (T1-R, T2-R, T3-R) in medians arederived. The distribution of T_(50%)-GIR-AUC₀₋₃₆ values is representedby histogram plots for each treatment. In addition, a histogram ofdifferences in T_(50%)-GIR-AUC₀₋₃₆ between treatments (T1-R, T2-R, T3-R)is provided.

The distribution of GIR-T_(max) values is represented by histogram plotsfor each treatment. In addition, a histogram of differences inGIR-T_(ma), between test treatments and reference is provided.

Duration of euglycemia and of blood glucose control are presented byhistogram plots.

Treatment comparisons are performed non-parametrically.

Performance of Clamp

Individual profiles of blood glucose concentration are plotted.

Duration of clamp is derived per clamp as the time between dosing andend of clamp in hours.

Individual variability of blood glucose per clamp is derived as thecoefficient of variation (CV %) of blood glucose values betweenindividual start and individual end of clamp (or first administration ofinsulin glulisine during clamp). Individual average blood glucose levelper clamp is derived as the arithmetic mean of blood glucose valuesbetween individual start and individual end of clamp (or firstadministration of insulin glulisine during clamp).

Parameters are listed individually and summarized descriptively withintreatment.

Analysis of Safety Data

The safety evaluation is based upon the review of the individual values(potentially clinically significant abnormalities), descriptivestatistics (summary tables, graphics) and if needed on statisticalanalysis (appropriate estimations, confidence intervals). “PotentiallyClinically Significant Abnormalities” (PCSA) criteria are used accordingto standard criteria of sanofi-aventis. Criteria are documented in thestatistical analysis plan of this study. The safety analysis isconducted according to the sanofi-aventis standards related to analysisand reporting of safety data from clinical trials.

All safety analyses encompass data of the safety population.

For all safety data, the observation period is divided into segments ofthree different types:

-   -   the pre-treatment period is defined as the time between when the        subject gives informed consent and the first administration of        study medication.    -   the on-treatment period is defined as the time from (first)        study medication administration up to 72 hours later.    -   the post-treatment period is defined as the time after        on-treatment period to either the (first) administration of        study medication in the next period or the end of the follow-up        period.

Adverse Events

All AEs are coded using MedDRA (latest version in use at time ofdatabase lock).

The following listings are provided for all adverse events:

-   -   Listing of all adverse events (by subject)    -   Listing of comments related to adverse events

Definitions

For safety data, the observation period is divided into segments ofthree different types:

-   -   the pre-treatment period is defined as the time between when the        subject gives informed consent and the first administration of        comparative study medication.    -   the on-treatment period per period is defined as the time from        (first) study medication administration up to 72 hours later.    -   the post-treatment period is defined as the time after        on-treatment period to either the (first) administration of        study medication in the next period or the end of the follow-up        period.

Treatment Emergent Adverse Events

All AEs are classified as follows:

-   -   Treatment-emergent adverse events (TEAEs) are any AEs with an        onset (incl. worsening) during an on-treatment period    -   Non-treatment-emergent adverse events (NTEAEs) are any AEs not        classified as TEAE:        -   Pre-treatment AEs, defined as AEs that developed (or            worsened) during the pre-treatment period before the first            dose of study medication        -   Post-treatment AEs, defined as AEs that developed during a            post-treatment period without worsening during an            on-treatment phase.

Assignment to Treatments

For analysis purposes, each TEAE is assigned to the last treatment givenbefore onset (or worsening) of the AE. If a TEAE develops on onetreatment and worsens under a later treatment, it is consideredtreatment emergent for both treatments.

Missing Information

In case of missing or inconsistent information, an AE is counted as aTEAE, unless it can clearly be ruled out that it is not a IBAE (e. g. bypartial dates or other information).

If the start date of an AE is incomplete or missing, it is assumed tohave occurred after the first administration of study medication exceptif an incomplete date indicates that the AE started prior to treatment.

Treatment-Emergent Adverse Events

Treatment emergent adverse events are listed and summarized bytreatment:

-   -   Overview of TEAEs (number and percentage of subjects with at        least one TEAE, severe TEAE, TEAE leading to discontinuations,        death (if any))    -   Summary of all treatment-emergent adverse events by primary        system organ class and preferred term (number and percentage of        subjects with at least one TEAE) (“in-text table”)        -   Table without number of events (for body of the clinical            study report)        -   Table with number of events (for appendix of the clinical            study report)        -   Table with number of subjects per formulation (U100, U300)            and of subjects overall (for appendix of the clinical study            report)    -   Listing of subjects presenting treatment emergent adverse events        by treatment, system organ class and preferred term

Deaths, Serious and Other Significant Adverse Events

In case of any occurrences, deaths, serious AEs, and other significantAEs are listed individually and described in the study report in detail.

Adverse Events Leading to Treatment Discontinuation

In case of any occurrences, individual subject listings are generatedfor all adverse events leading to treatment discontinuation.

Clinical Laboratory Evaluations Hematology and Biochemistry Data

Laboratory safety parameters are measured on D1 of treatment period 1and at EOS. Per schedule, these safety parameters are assessed duringthe on-treatment period (except hematology at TP3 and TP4).

The values to be used as baseline (hematology and biochemistry) are thevalues collected on D1 predose in the first treatment period. If any ofthe scheduled baseline tests are repeated for any subject, the lastrechecked values are considered as baselines, provided they were donebefore the first IP administration.

The following tables and listings are provided:

-   -   Descriptive statistics for raw data and changes from baseline        (including % change for creatinine)    -   A specific listing of individual data from subjects with        post-baseline PCSAs will be provided, sorted by function and        time of measurement    -   All individual data, including rechecked values, for planned        hematology and biochemistry, are listed by biological function        and time of measurement. If any, data from unscheduled        laboratory tests are included in this listing. In these        listings, individual data are flagged when lower or higher than        the lower or upper laboratory limits and/or when reaching the        absolute limit of PCSA criteria, when defined    -   A listing of liver function data for subjects, who experienced        at least one of the following:        -   at least one occurrence of ALT>3ULN and at least one            occurrence of total bilirubin            -   >2 ULN during the study with at least one of them being                post first dose        -   conjugated bilirubin>35% total bilirubin and total            bilirubin>1.5 ULN will be provided on the same sample post            first dose, irrespective of the definition for the            on-treatment phase.    -   A listing related to increase in ALT≥2 ULN is provided,        including notably the information on drug intake, medical and        surgical history, alcohol habits, trigger factors, event details        with ALT values, associated signs and symptoms.    -   A listing of out-of-range definitions is provided.

In the listings of subjects with PCSAs, liver function data, CPK, andeosinophils are expressed as multiple of the corresponding ULN.

Urinalysis Data

All qualitative urinary test results (dipstick), including recheckedvalues, are listed.

Vital Signs Blood Pressure and Heart Rate

Heart rate and systolic and diastolic blood pressure (SBP and DBP) aremeasured after 10 minutes in supine resting position and also after 3minutes in standing position, except when connected to the Biostator™.

The values to be used as the baselines are the D1 pre-dose assessmentvalue of each treatment period. If any of the scheduled baseline testsare repeated for any subject, the last rechecked values are consideredas baselines, provided they were done before the IP administration.

For heart rate and blood pressures, orthostatic differences arecalculated as the change from supine to standing position.

For all parameters, an “On-Treatment” analysis will be performedincluding all unplanned values and rechecked values.

The following tables and listings are provided:

-   -   Summary tables of counts of subjects with PCSAs are provided as        incidence tables of post-baseline PCSAs, regardless of the        normal or abnormal status of the baseline    -   For heart rate and blood pressures (supine and standing        positions), raw data and changes from baseline (supine position        only) are summarized in descriptive statistics, for type of        measurement (position) each parameter and time point, based on        planned pre-dose measurements and the baseline defined    -   All individual data, including unplanned and rechecked values,        are listed (supine, standing, orthostatic difference). In the        listings, values are flagged when reaching the limits of the        PCSA criteria when defined    -   A data listing of individual post-baseline PCSAs is provided    -   Comments related to vital sign evaluations are also listed in        the Appendix, if any.

Body Weight, Body Mass Index, and Body Temperature

The values to be used as baselines for body weight and BMI are thevalues collected on D1 of TP1.

The values to be used as baselines for body temperature are the valuescollected on D1 of each TP.

Individual data are listed including flags (weight only) for values whenreaching the limits of the PCSA criteria.

ECG

Heart rate, PQ-, QRS-, and QT-intervals and corrected QT (QTc) fromautomatic reading are analyzed as raw parameter value and change frombaseline.

The values to be used as the baseline are the Day 1 predose value ofeach period. If any of the scheduled baseline tests are repeated for anysubject, the rechecked values are considered as baselines, provided theywere done before the drug administration of the period.

For all parameters, an on-treatment analysis is performed using allpost-baseline assessments done during the on-treatment period, includingrechecked values. Counts of subjects with postbaseline PCSAs areprovided in summary tables regardless of the normal or abnormal statusof the baseline, by treatment group.

Raw data for all parameters and change from baseline are summarized indescriptive statistics by parameter, treatment, and time of measurement.

Individual data, including rechecked values, are listed, sorted bytreatment, subject, visit and time of measurement. In the listings,values reaching the limits of the PCSA criteria are flagged.

A listing of individual data from subjects with post-baseline PCSAs isprovided, sorted by type of measurement and sorted by subject, period,and time of measurement.

Additionally, a separate listing of the cardiac profile for subjectswith prolonged QTc (>450 ms for Males and >470 ms for Females) orchanges from baseline in QTc>60 ms (for males and females) and a listingof subjects with at least one abnormality in qualitative assessment(i.e., abnormal ECG) after the 1st dosing are also provided.

Other Related Safety Parameters Physical Examination

Listing of comments related to physical examination is provided, if any.

Local Tolerability at Injection Site

Frequency distributions by treatment are provided for levels of localtolerability at injection site. Individual data are listed. Within eachcriterion and treatment, a subject is counted with their most severeresult.

Allergic Reactions Listings for Allergic Reactions

Any cases of allergic reaction are documented as adverse events withdetailed complementary information. All cases are described in detail inthe clinical study report.

Individual cases and all complementary data are listed.

Allergic Medical History and Family Medical History

Allergic medical history and family medical history is documented forsubjects with any occurrence of potential allergic reaction. All detailsof allergic medical history and of allergic family medical history arelisted on an individual basis.

Anti-Insulin Antibodies

A summary table is provided with the number of subjects for theanti-insulin antibodies results during the study and from the post studyinvestigations. Individual subject listing is provided.

Analysis of Pharmacokinetic Data Pharmacokinetic Parameters

The list of PK parameters is shown above. In addition, T_(50%)-AUC₀₋₃₆for insulin is derived in the context of the statistical analysis.

Statistical Analysis

Pharmacokinetic parameters of insulin glargine are listed and summarizedusing at least arithmetic and geometric means, standard deviation (SD),standard error of the mean (SEM), coefficient of variation (CV %),minimum, median and maximum for each treatment.

All pharmacokinetic analyses encompass data of the correspondingpharmacokinetic populations as defined above. No adjustment of thealpha-level is made for multiple analyses.

Statistical analyses compare test treatments (T₁ to T₃) versus referencetreatment (R).

Analysis of Treatment Ratios

The analysis is performed for AUC₀₋₃₆ for insulin glargine. Prior to allanalysis described below, AUC₀₋₃₆ values are log-transformed (naturallog).

Log-transformed parameters are analyzed with a linear mixed effectsmodel with fixed terms for sequence, period and treatment

log(parameter)=sequence+period+treatment+error,

and with an unstructured R matrix of treatment (i, i) variances andcovariances for subject within sequence blocks, using SAS PROC MIXED.

Estimate and 90% confidence interval (CI) for the ratio of treatmentsgeometric means (T₁/R, T₂/R, T₃/R) are obtained by computing estimateand 90% CI for the difference between treatment means within the linearmixed effects model framework, and then converting to ratio of geometricmeans by the antilog transformation. Bioequivalence is concluded if the90% CI for the ratio is entirely within the 0.80 to 1.25 equivalencereference interval.

Listings of individual treatment ratios (T₁/R, T₂/R, T₃/R) are providedwith the corresponding descriptive statistics.

T_(50%)-AUC₀₋₃₆ for insulin

The distribution of T_(50%)-AUC₀₋₃₆ values for insulin is represented byhistogram plots for each treatment. In addition, a histogram ofdifferences in T_(50%)-AUC₀₋₃₆ between treatments (T₁-R, T₂-R, T₃-R) isprovided.

T_(50%)-AUC₀₋₃₆ (h) is analyzed non-parametrically.

Dose Exposure Relationship for Insulin Glargine U300 DescriptiveAnalyses of Dose Exposure Relationship

Dose exposure relationship for insuline glargine U300 is describedgraphically by

-   -   plots per subject of exposure over total dose per subject    -   plots per subject of exposure over dose per kg bodyweight    -   plots per subject of dose normalized exposure over dose per kg        bodyweight (dose normalization on 0.6 U/kg)

If deemed necessary for interpretation of results, additionaldescriptive analyses are added.

Statistical Analysis of Dose Exposure Relationship

For AUC of insulin glargine calculated for the test treatments T₁-T₃,dose exposure relationship is assessed using the empirical power model(PK-parameter=a*dose^(b)), along with an “estimation” interpretation,according to the recommendations in Gough et al. (Gough K, Hutchison M,Keene O et al. Assessment of dose proportionality: report from thepharmaceutical industry. Drug Information Journal 1995; 29:1039-1048).

The empirical power model provides a readily and interpretable measureof the degree of non-proportionality, which can be used both to confirmproportionality and to assess the pharmacokinetic and clinicalsignificance of any departures. The analysis of dose proportionalitystudies, however, requires estimation rather than significance testingin order that the pharmacokinetic and clinical significance of anynon-proportionality can be assessed.

The power model is fit on the log-transformed scale using a randomcoefficients power model for dose (in U/kg body weight):

log(parameter)=(log(alpha)+alpha[i])+(beta+beta[i])*log(dose)

where log(alpha) and beta are the population intercept and slope,respectively, and alpha[i] and beta[i] are the random deviations fromalpha and beta, respectively, for the i-th subject.

Estimates for beta with 90% confidence intervals are obtained viaestimated generalized least squares in the SAS®/PROC MIXED procedure,with restricted maximum likelihood (REML) estimates of covarianceparameters. Estimates and 90% confidence intervals for beta are furtherused to obtain estimates and 90% confidence intervals for the PKparameter increase associated with an r-fold increase in dose (r=1.5 andr=2.25 [i. e. high dose/low dose]), by exponentiating r to the powers ofthe beta estimate and confidence limits.

If there is evidence of model lack-of-fit, the mixed effect model (asused for analysis of treatment ratios) is used for the analysis.Estimates with 90% CIs for the parameter increases associated withpairwise dose increases are obtained by first computing estimates withCIs for pairwise differences between doses in the mixed effects modelframework, and then converting to ratios using the antilogtransformation.

PK/PD Analysis

If appropriate, graphical displays (scatter plots) are generated toexplore PK/PD relationship.

Example 14: Study Results Subject Disposition

A total of 24 subjects with Type 1 diabetes mellitus were enrolled,randomized and received at least one dose of study medication. Of the 24randomized subjects, 2 subject withdrew from the study on own request.Twenty-two (22) subjects completed the study according to the protocoland were included in the pharmacodynamic (PD) and pharmacokinetic (PK)analyses. All 24 treated subjects were included in the safetyevaluation.

There were no major protocol deviations.

Demographics Characteristics

The following data (Table 12) were collected: sex, age at screening,height, weight, and race. Body mass indexes (BMI) per subject werecalculated from body weight and height data: BMI=body weight[kg]·(height [m])⁻².

TABLE 12 Demographics Sex BMI (kg/m²) Weight (kg) Age (years) Race (n)[%] N 5 F, 25.55 79.38 42.6 Caucasian/ N 24 19 M 1.99 (SD) 9.67 (SD)10.0 (SD) white 24 min 20.5: min 57.3: min 19: [100] max 28.3 max 94.3max 60

Clamp Performance

At the four treatment periods for each subject, R (Lantus U100), T1 (0.4U/kg HOE901-U 300), T2 (0.6 U/kg HOE901-U 300) and T3 (0.9 U/kg HOE901-U300), the individuals' baseline blood glucose concentrations prior toinsulin medication were similar, defining the clamp level at 100 mg/dL.The duration of the observation period of the clamps after dosing was 36hours and the same in all treatment periods.

Primary Endpoints

Equivalence in bio-availability (exposure) and bio-efficacy (activity)for R and T was not established.

Primary Variables

The area under the serum insulin glargine concentration time curve from0 to 36 hours (INS-AUC_((0-36h))) was not equivalent for R and T1 and T2and about equivalent with T3. The exposure was estimated to be less byabout 37% with T1, less by about 43% with T2 and similar with T3,compared to R.

The area under the GIR versus time curve from 0 to 36 hours(GIR-AUC₍₀₋₃₆₁₀₎ was not equivalent for R and T1 and T2 and aboutequivalent with T3. The exogenous glucose consumption required topreserve blood glucose control was estimated to be less by about 88%with T1, 67% with T2 while about similar with T3.

Secondary Variables

The time to 50% of INS-AUC_((0-36h)) (h) with R was about 14 h and thusshorter as compared to about 16 h, 16 h and 19 h with T1, T2 and T3,respectively.

The time to 50% of GIR-AUC_((0-36h)) (h) with R was about 12 h and thusshorter as compared to about 17 h, 18 h and 20 h with T1, T2 and T3,respectively.

Safety

No serious adverse events (AEs) or withdrawals due to AEs were reported.Two subjects on R, 2 on T1 and 4 on T3 reported a total 8 TEAEs, all ofwhich were of mild to moderate intensity, and resolved without sequalae.The most frequently reported event was headache. Of note, headache is acommon observation for clamp studies and is related to the infusion ofhyper-osmolaric glucose solutions. However, a link to theinvestigational products cannot be excluded. No injection site reactionswere reported with T1, T2 and T3 while 2 subjects on R developed hardlyperceptible erythema at the injection site.

Conclusions

Same doses of R and T U 300 are not equivalent in bio-availability(exposure) and bio-efficacy (activity) after single dose administration.Exposure and activity after T1 (0.4 U/kg) and T2 (0.6 U/kg) were less ascompared to exposure and activity after administration of R (0.4 U/kg).R and T3 were virtually equivalent as to exposure and exogenous glucoseconsumption.

T1, T2 and T3 did, however, show yet flatter PK (exposure) and PD(activity) profiles with even less fluctuation around the averages thanR, i.e., a profile as it would be desired for basal insulin supply. Thisis particularly evident when comparing R and T3 which provide nominalequivalent total exposure and total glucose consumption though ofdifferent profiles.

These surprising and unexpected differences in exposure and activitybetween R (Lantus U100) and T (HOE901-U300) formulations in subjectswith type 1 diabetes mellitus are effectively shown in the figuresbelow.

Over and above, administration of T (HOE901-U300) was without safety andtolerability issues.

Example 15: Study Rationale for Comparing the Glucodynamic Activity andExposure of Two Different Subcutaneous Doses of (HOE901-U300) to LantusU100 in Patients with Type 1 Diabetes Mellitus

Results from the study in healthy subjects and in subjects with Type 1diabetes mellitus (see foregoing examples) showed exposure andeffectiveness not to be equivalent between Lantus® U100 and insulinglargine U300. Subjects received the same dose of insulin glargine (0.4U/kg) for U100 and U300, but delivery of the same unit-amount from U300produced less exposure at less exogenous glucose consumption to preserveblood glucose control than delivery from U100. Though Lantus U100 showsexposure and pharmacodynamic profiles without pronounced fluctuationaround the averages, HOE901-U300 did, however, show even lessfluctuation in exposure and pharmacodynamic profiles, as it would bedesired for basal insulin supply, with a yet even longer duration ofaction.

In order to assess the pharmacokinetic and pharmacodynamic profile understeady state conditions, a new study described in the following examplestherefore compares two different subcutaneous doses of insulin glargineU300 versus a standard dose of Lantus® U100 as comparator with a finaleuglycemic clamp setting in patients with type 1 diabetes mellitus. Thisstudy aims to estimate an U300 dose that is equieffective to 0.4 U/kgLantus® U100 as assessed by parameters of blood glucose control andblood glucose disposal provided by the clamp technique.

Insulin glargine exposure is assessed from concentration-time profilesafter repeated subcutaneous administration at steady state, and activityas glucose utilization per unit insulin at steady state.

The study comprises two cross-over treatments (R and T1, and R and T2)in 2 parallel groups, with 2 treatment periods (TP1, TP2) and 2sequences, each. There are one screening visit (D-21 to D-3), treatmentvisits (D1 to D10 in TP1 and TP2 with evening dosing), with in-houseperiods (D1 to D4 morning and D8 morning to D10 evening for clampassessments) and one end-of-study visit (between D7 to D10 after lastdosing) with final assessment of safety parameters.

The Lantus® U100 dose of 0.4 U/kg selected for the study is wellcharacterized to provide euglycaemic blood glucose control in type 1diabetes patients and has been readily investigated in other clampstudies with type 1 diabetes patients.

Two different doses are tested for insulin glargine U300, 0.4 and 0.6U/kg. This dose range allows intrapolating an approximate doseequieffective to 0.4 U/kg Lantus® U100. The dose of 0.4 U/kg of insulinglargine U300 has already been tested in healthy volunteers and subjectswith type diabetes mellitus (see foregoing examples) and was found to beless active than 0.4 U/kg Lantus® U100 within 30 and 36 hours,respectively, the predefined ends of the observation periods. Bloodglucose control with 0.4 U/kg insulin glargine U300 required less totalglucose disposition than that of reference medication (0.4 U/kg Lantus®U100). A correspondingly higher dose of insulin glargine U300, e.g. 0.6U/kg insulin glargine U300, is expected to result in even tighter bloodglucose control at less total glucose disposition. Moreover, theproportional dose escalation allows exploring exposure and effectprofiles for dose-proportionality.

A study in patients with type 1 diabetes avoids confounding impact ofendogenous insulin and better permits assessment of exposure andduration of action.

This study has a cross over design; based on the outcome of previousstudies not more than two HOE901-U300 doses will be compared to Lantus®U100. Assessment of glucodynamic activity of long acting insulinproducts requires a euglycemic clamp setting beyond 24 hours, thepredefined injection interval, owed to the extended duration of action.

The active pharmaceutical ingredient, insulin glargine, is the same inboth formulations, U100 and U300. The doses used in this study arewithin the range of regular use. Although an overall risk ofhypoglycemia is not completely excluded, it is controlled by theeuglycemic clamp technique.

Pharmacodynamics

The pharmacodynamic activity of insulin glargine is evaluated by theeuglycemic clamp technique in type 1 diabetes patients, which is theestablished standard procedure to evaluate the effect of exogenousadministered insulin products on blood glucose disposal.

Parameters specific for assessment of glucose disposition in aeuglycemic clamp setting are the body weight standardized glucoseinfusion rate (GIR), total glucose disposed within 24 and 36 hours,respectively, GIR-AUC₀₋₂₄ and GIR-AUC₀₋₃₆, and times to a givenpercentage of GIR-AUC₀₋₂₄ and GIR-AUC₀₋₃₆ such as time to 50% ofGIR-AUC₀₋₃₆.

Ancillary parameters are the maximum smoothed body weight standardizedGIR, GIR_(max), and Time to GIR_(max), GIR-T_(max).

Duration of action of insulin glargine is derived from the time betweendosing and pre-specified deviations above the euglycemic (clamp) level.

Glucose monitoring is performed for 36 hours due to the long duration ofaction of insulin glargine after subcutaneous administration

Pharmacokinetics

Due to the sustained release nature of insulin glargine there is a lackof pronounced peaks in the concentration profile. Therefore, the time to50% of INS-AUC (e.g. T_(50%) INS-AUC₀₋₃₆) is calculated as a measure forthe time location of the insulin glargine exposure profile, andINS-C_(max) and INS-T_(max) will serve as additional measures.

Primary Study Objectives

The primary objective of the study is to assess the blood glucosecontrol and the required exogenous glucose consumption of two differentinsulin glargine U300 doses versus 0.4 U/kg Lantus® U100 in steadystate.

Secondary Study Objectives

The secondary objectives of the study are to assess in steady state, theexposure ratios of two different insulin glargine U300 doses versus 0.4U/kg Lantus® U100, to compare the duration of action of two differentinsulin glargine U300 doses versus 0.4 U/kg Lantus® U100, to explore thedose response and dose exposure relationship of insulin glargine U300,and to asses the safety and tolerability of insulin glargine U300 insubjects with type 1 diabetes.

Example 16: Change of Dissolution Properties of Acidic Formulations ofLong-Acting Insulins at Higher Concentrations

The influence of the higher concentrations of insulin glargineformulations with regard to dissolution properties are investigated byusing an in-vitro test system. To do so, precipitation studies areperformed using a phosphate buffer with a pH of 7.4, simulating thein-vivo conditions.

The supernatant of the precipitated insulin is investigated using HPLCtechnique to determine the insulin glargine content.

Detailed Description of the Studies: Preparation of the PrecipitationBuffer Solution:

19.32 mg sodium dihydrogen phosphat monohydrat (M: 137.98 g/mol) aredissolved per mL water. 0.1 M sodium hydroxide or 0.1 M hydrochloricacid is used for adjustment of the pH to 7.4.

Performance of the Precipitation Studies:

Solutions of insulin glargine drug product having concentrations of upto 1000 U/mL and comprising the same total amount of insulin glargineand the buffer are placed in plastic tubes and are slightly shaken.After precipitation of the insulin glargine the dispersions arecentrifuged at slow rotations for a pre-defined time period. A definedvolume of the dissolution medium is taken out and replaced with freshbuffer medium.

Determination of the Insulin Content:

The content of insulin glargine in the samples from the supernatant isquantified against the respective insulin reference standard byreverse-phase-HPLC using a two mobile phase system, containing a sodiumdihydrogenphosphate buffer in water, sodium chloride (NaCl) anddifferent amounts of acetonitrile.

As stationary phase an octadodecyl-column is used, detection wavelengthis 215 nm.

The release profile of insulin glargine from the higher concentratedsolutions (e.g. U500 and U1000) is flatter and prolonged compared toLantus U100.

Example 17: Microscopic Investigation of Precipitates

The precipitates of insulin glargine formulations having concentrationsof 100 U/mL, 300 U/mL, 500 U/mL 700 U/mL and 1000 U/mL have beeninvestigated by microscopy. Said formulations (with an identical amountof 60 U of insulin glargine) have been precipitated in 200 μL of aphosphate buffer, pH 7.4 and were investigated by transmitted lightoptical microscope (Olympus Model BX61) with the magnitudes 100×, thepictures are shown in the following also presenting the maximumdiameters. These investigations revealed differences in theprecipitations characteristics, leading to remarkable bigger particleswith increasing concentrations. The results are shown in FIGS. 8A-8E.

Example 18: Blood Glucose Lowering Effect of Insulin Glargine in Dogs

The blood glucose lowering effect of insulin glargine was evaluated inhealthy, normoglycemic Beagle dogs. The dogs received singlesubcutaneous injections of 0.3 IU/kg. Venous blood glucose wasdetermined before the first injection and subsequently up to 24 h.

Animals were taken from cohort of −30 healthy, normoglycemic male Beagledogs, originally obtained from Harlan. The dogs were maintained inkennel groups under standardized conditions. The day before study startthe dogs were randomly distributed to study cages. They were fasted 18hours prior to start and throughout the experiment with free access totap water. Body weight of the dogs in the present study was between 13and 27 kg. After each experiment the dogs were allowed to recover for atleast two weeks.

The animals were randomized to groups of n=6. At time point zero theanimals were treated with single doses of the test compound. Insulinglargine was administered as a single subcutaneous injection a dose of0.3 IU/kg.

Blood sampling was performed consecutively via puncture of the forearmvein (Vena cephalica) before drug administration (0 h) and thereafter upto 24 hours. Blood glucose was determined enzymatically (Gluco-Quant®Glucose/HK kit on Roche/Hitachi 912).

The effect on blood glucose following subcutaneous injection ofdifferently concentrated preparations of insulin glargine, 100 and 300units/mL, was tested in healthy, normoglycemic Beagle dogs

With increasing insulin glargine concentration the mean time of actionincreased from 6.8h (U100) to 7.69h (U300), respectively.

By increasing the glargine concentration from 100 to 300 U/mL the bloodglucose decreasing time-action profile was changed towards a flatter andprolonged activity in the dog

The current data in dogs is consistent with data in humans showing thathigher drug concentrations of insulin glargine are positively correlatedwith profile and longer duration of action.

LIST OF ABBREVIATIONS ° C. Degrees Celsius ABE Average Bioequivalence AEAdverse Event ALT Alanine Aminotransferase

aPPT activated Partial Thromboplastin Time

ARF Acute Renal Failure AST Aspartate Aminotransferase β-HCG Beta-HumanChoriongonadotropine

bpm beats per minutecm centimeter

CPK Creatinine Phosphokinase CRF Case Report Form DRF DiscrepancyResolution Form ECG Electrocardiogram

EOS End-of-study (visit)

GCP Good Clinical Practice

GGT Gamma-glutamyl transferase

Hb Hemoglobin

HbA1c Glycocylated hemoglobinHBs Hepatitis B surface

Hct Hematocrit HCV Hepatitis C Virus HIV Human Immunodeficiency Virus HRHeart Rate INN International Nonproprietary Name

INR International Normalized Ratio (prothrombin time)

IP Investigational Product IRB/IEC Institutional ReviewBoard/Independent Ethics Committee Kg Kilogram

LOQ Limit of quantification

PT Prothrombin Time

QTc QT interval automatically corrected by the ECG machineQTcB QT interval corrected by Bazett formulaQTcF QT interval corrected by Fridericia formulaQtcN QT interval corrected by a population approachQtcNi QT interval corrected by individual population approachRBC Red Blood Cell count

SBP Systolic Blood Pressure

SCR Screening (visit)

UDS Urine Drug Screen

ULN Upper Limit of Normal rangeWBC White Blood Cell count

1. An aqueous pharmaceutical formulation comprising 200-500 U/mL ofinsulin glargine [equimolar to 200-500 IU human insulin].
 2. The aqueouspharmaceutical formulation of claim 1 comprising 200 U/mL of insulinglargine [equimolar to 200 IU human insulin].
 3. The aqueouspharmaceutical formulation of claim 1 comprising 500 U/mL of insulinglargine [equimolar to 500 IU human insulin].
 4. The aqueouspharmaceutical formulation of claim 1 further comprising at least oneexcipient selected from the group consisting of zinc, m-cresol,glycerol, and polysorbate
 20. 5. The aqueous pharmaceutical formulationof claim 4 further comprising 90 μg/mL zinc, 2.7 mg/mL m-cresol, and 20mg/mL glycerol 85%.
 6. The aqueous pharmaceutical formulation of claim5, wherein the zinc is present in the form of zinc chloride.
 7. Theaqueous pharmaceutical formulation of claim 1, wherein the pH of theaqueous pharmaceutical formulation is between 3.4 and 4.6.
 8. Theaqueous pharmaceutical formulation of claim 7, wherein the pH of theaqueous pharmaceutical formulation is 4.0.
 9. An aqueous pharmaceuticalformulation comprising 200 U/mL of insulin glargine [equimolar to 200 IUhuman insulin] wherein the pH of the aqueous pharmaceutical formulationis between 3.4 and 4.6.
 10. The aqueous pharmaceutical formulation ofclaim 9, wherein the pH of the aqueous pharmaceutical formulation is4.0.
 11. The aqueous pharmaceutical formulation of claim 9 furthercomprising at least one excipient selected from the group consisting ofzinc, m-cresol, glycerol, and polysorbate
 20. 12. The aqueouspharmaceutical formulation of claim 11 further comprising 90 μg/mL zinc,2.7 mg/mL m-cresol, and 20 mg/mL glycerol 85%.
 13. The aqueouspharmaceutical formulation of claim 12, wherein the zinc is present inthe form of zinc chloride.
 14. The aqueous pharmaceutical formulation ofclaim 1, wherein the pharmacokinetic profile and the pharmacodynamicprofile of the aqueous pharmaceutical formulation followingadministration to a patient are flatter than the pharmacokinetic profileand the pharmacodynamic profile of an aqueous pharmaceutical formulationcomprising 100 U/mL of insulin glargine, and wherein the pH of theaqueous pharmaceutical formulation is between 3.4 and 4.6.
 15. Theaqueous pharmaceutical formulation of claim 14, wherein the pH of theaqueous pharmaceutical formulation is 4.0.
 16. The aqueouspharmaceutical formulation of claim 14 further comprising at least oneexcipient selected from the group consisting of zinc ion, m-cresol,glycerol, and polysorbate
 20. 17. The aqueous pharmaceutical formulationof claim 16 further comprising 90 μg/mL zinc, 2.7 mg/mL m-cresol, and 20mg/mL glycerol 85%.
 18. The aqueous pharmaceutical formulation of claim17, wherein the zinc is present in the form of zinc chloride.
 19. Theaqueous pharmaceutical formulation of claim 1, wherein the aqueouspharmaceutical formulation provides a peakless long acting basalinsulin, and wherein the pH of the aqueous pharmaceutical formulation isbetween 3.4 and 4.6.
 20. The aqueous pharmaceutical formulation of claim1, wherein the aqueous pharmaceutical formulation exhibits an extendedduration of exposure of insulin glargine than an aqueous pharmaceuticalformulation comprising 100 U/mL insulin glargine, and wherein the pH ofthe aqueous formulation is between 3.4 and 4.6.